Tagungsprogramm Abstracts 6. - 9. März 2005 38. Diskussionstagung ...

Deutsche Gesellschaft für

Massenspektrometrie

38. Diskussionstagung 6. - 9. März 2005 Rostock Tagungsprogramm Abstracts

Proteom-Zentrum Rostock Universität Rostock

DGMS2005

38. Diskussionstagung der Deutschen Gesellschaft für Massenspektrometrie 6. – 9. März 2005

Rostock

Tagungsprogramm Abstracts der Vorträge und Poster

Proteom-Zentrum Rostock Universität Rostock

Impresssum

Herausgeber / Editor: Proteom-Zentrum Rostock Abteilung für Proteomforschung Institut für Immunologie Medizinische Fakultät Universität Rostock Joachim-Jungius-Str. 9 18059 Rostock Deutschland

Redaktion / Compilation: Cornelia Koy Michael Kreutzer Michael O. Glocker

Druck / Print: Schaffarzyk Die Agentur Die Druckerei Strandstraße 17a 18225 Kühlungsborn Germany

ISBN 3-00-015648-8

Dieses Werk ist urheberrechtlich geschützt. Die dadurch begründeten Rechte, insbesondere die der Übersetzung, des Nachdrucks, der Entnahme von Abbildungen, der Wiedergabe auf photomechanischem oder ähnlichem Wege und der Speicherung in Datenverarbeitungsanlagen bleiben – auch bei nur auszugsweiser Verwendung – vorbehalten. Die Autoren übernehmen die Verantwortung für den Inhalt der Abstracts.

Werbeseite 1 (GSG)

Wissenschaftliches Programmkomittee / Scientific Committee Prof. Dr. Lutz Schweikard Prof. Dr. Karl-Heinz Meiwes-Broer Prof. Dr. Michael O. Glocker Dr. Joachim Wesener

Lokales Organisationskomittee / Local Organizers Prof. Dr. Lutz Schweikard Prof. Dr. Karl-Heinz Meiwes-Broer Prof. Dr. Michael O. Glocker Dr. Cornelia Koy

Inhalt / Content

GRUßWORT / PREAMBLE

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ÜBER DAS PROTEOM-ZENTRUM ROSTOCK / ABOUT THE PROTEOME CENTER ROSTOCK

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VERANSTALTUNGSSTANDORTE UND TAGUNGSRÄUME / LOCATIONS AND CONFERENCE ROOMS

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DANKSAGUNG / ACKNOWLEDGEMENTS

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TAGUNGSPROGRAMM – ÜBERSICHT / PROGRAM OVERVIEW

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WORKSHOPS

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HAUPTVORTRAGENDE / PLENARY LECTURERS

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TAGUNGSPROGRAMM / SCIENTIFIC PROGRAM

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LECTURES / WORKSHOPS: SUNDAY, 6TH OF MARCH, 2005

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POSTERS: MONDAY, 7TH OF MARCH, 2005 LECTURES: MONDAY, 7TH OF MARCH, 2005

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POSTERS: TUESDAY, 8TH OF MARCH, 2005 LECTURES: TUESDAY, 8TH OF MARCH, 2005

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LECTURES: WEDNESDAY, 9TH OF MARCH, 2005

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ABSTRACTS

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AUTORENVERZEICHNIS / AUTHOR INDEX TEILNEHMERVERZEICHNIS / LIST OF PARTICIPANTS

Grußwort / Preamble Grußwort Das Proteom-Zentrum Rostock und die neu gegründete Abteilung für Proteomforschung des Instituts für Immunologie der Universität Rostock freuen sich über die Wahl der Deutschen Gesellschaft für Massenspektrometrie, die 38. Diskussionstagung in diesem Jahr in Rostock auszutragen und insbesondere darüber, uns als lokalen Organisator mit dieser ehrenvollen Aufgabe zu betrauen. Die Jahrestagung der DGMS hat in wissenschaftlichen Kreisen dank des hervorragenden Programms seit vielen Jahren ein international hoch anerkanntes Renommee, das auch in diesem Jahr beispielsweise durch die zunehmende Teilnahme nationaler und insbesondere internationaler Teilnehmer unterstrichen wird. Folgerichtig hat die Gesellschaft im Einvernehmen mit den lokalen Veranstaltern entschieden, dass deutsch und englisch gleichberechtigte Tagungssprachen sein werden. In diesem Jahr werden wir wegen der erfreulicherweise sehr zahlreichen Zusendungen von Abstracts erstmals Parallel-Vorträge durchführen. Dies ist auch deshalb möglich, weil die naturwissenschaftliche Fakultät der Universität Rostock uns großzügigerweise gleich zwei Hörsaalzentren für unsere Tagung zur Verfügung stellt, wofür wir uns an dieser Stelle im Namen der Gesellschaft sehr herzlich bedanken möchten. Wir freuen uns sehr, dass wir somit auch in der Lage sind, dem Engagement der Firmen durch gesonderte Firmenpräsentationen im entsprechenden Rahmen im Tagungsprogramm entsprechen zu können. Ein Novum ist außerdem, die Vormittagspräsentationen am Montag und Dienstag mit den Posterdarbietungen zu beginnen. Die Idee dahinter ist der Wunsch, den Posterpräsentatoren, ihrem herausragenden Stellenwert entsprechend, genügend Zeit zum Ideen- und Gedankenaustausch einzuräumen. Nicht unerwähnt bleiben soll das überaus erfreuliche Engagement der Firmen auch außerhalb des wissenschaftlichen Programms, das beispielsweise im Falle der Fa. Shimadzu dazu führt, dass der Tagungsband in der ansprechenden Form, wie Sie ihn in Händen halten, realisiert werden konnte. Danken möchte ich auch der Firma Waters für die Unterstützung des Konferenzdinners, der Firma Bruker für die Hilfe bei der Gestaltung des Begrüßungsabends, den Firmen Agilent und ABI Applied Biosystems für ihre Hilfe zu den Frühstückskaffees während der Posterdarbietungen, nicht zuletzt der Firma Thermo für die Zurverfügungstellung der Konferenztaschen und natürlich allen Firmen, die uns in großartiger Weise mit ihren Beiträgen unterstützen. Es ist das Ziel der Ausrichter, dass alle Veranstaltungsteile zusammen mit all Ihren Beiträgen und nicht zuletzt durch Ihre rege Teilnahme zu einer erfolgreichen Tagung führen sowie einen würdigen Rahmen für die diesjährigen Preisträger sowohl während der Verleihung der angesehenen Wissenschaftspreise der Gesellschaft als auch während der gesamten Tagung schaffen. Michael O. Glocker (Für das lokale Veranstaltungsteam der DGMS 2005)

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Preamble The Proteome Center Rostock and the newly founded Department for Proteome Research of the Institute of Immunology at the University of Rostock are delighted about the choice of the German Society for Mass Spectrometry, to arrange the 38th annual conference in Rostock and particularly to having consigned us as local organizers with this honorable assignment. The DGMS conference for many years has achieved an internationally recognized reputation in the scientific community due to its high-standard scientific program. This year´s conference reflects this reputation by the increasing participation of national and particularly international scientists. Hence, in accordance with the local organizers, the Society decided that both languages German and English shall be equally welcome as conference languages. For the first time we will meet in parallel sessions, due to the numerous abstracts that reached us for oral presentations. This endeavor has become feasible as the Faculty of Natural Sciences of the University of Rostock has generously opened even two lecture auditoriums to us, for which we want to express our cordial thanks in the name of the Society. We are happy that at the same time we will be able to adequately meet the commitment of the companies by introducing company presentations as a particular feature of our program. Beginning the morning presentations on Monday and Tuesday with the poster sessions is another novelty. The idea behind is the desire to cope with the significance of the poster presentations and to provide enough time for the exchange of thoughts and experience. We would like to emphasize the extraordinary dedication of the companies also outside of the scientific program that in the case of Shimadzu allowed to arrange the abstract book in that appealing way that you certainly notice holding the book in your hands. We also want to acknowledge Waters for their sponsorship of the conference dinner, Bruker for taking over the patronage of the welcome mixer, Agilent and ABI Applied Biosystems for taking responsibility for the poster breakfasts, Thermo for the beautiful conference bags, and last but not least all companies that support this conference in a bountiful way. It is the aim of the local organizers that all conference activities together with all your contributions and your lively attendance shall lead to a successful meeting and create a dignified ambience for this year´s laureates during the awards ceremony and during the entire conference. Michael O. Glocker (for the local organizing committee of the DGMS 2005)

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Über das Proteom-Zentrum Rostock / About the Proteome Center Rostock Über das Proteom-Zentrum Rostock Mit der Einrichtung des Proteom-Zentrums Rostock und der Berufung der ersten Professur für Proteomforschung in Deutschland (Prof. Glocker) wurde in Mecklenburg-Vorpommern mit Unterstützung des Bildungsministeriums, des Wirtschaftsministeriums, der Universität Rostock, der Stadt Rostock und der Steinbeis-Stiftung im Zuge einer „private-public partnership“ eine nachhaltige Forschungsstruktur geschaffen, die den Forschungsstandort Rostock im Bereich der Proteomforschung / Funktionelle Genomforschung und Bioinformatik mit engem Bezug zur klinischen Anwendung entscheidend prägt. In den vergangenen Jahren ist ein Zentrum mit überregionaler Ausstrahlung am Standort Rostock entstanden (s. z. B. Teilnahme von Herrn Nobelpreisträger Koichi Tanaka am 2. Rostocker Proteomforum im September 2003), wie auch der hohe Vernetzungsgrad in und außerhalb des Landes widerspiegelt. Die international ausgerichtete Tagung „2. Rostocker Proteomforum“ war Bestandteil des Programms „Jahr der Chemie“ 2003 des Bundesministeriums für Bildung und Forschung (BMBF). An der Universität Rostock wurde eine Abteilung für Proteomforschung gebildet, die das Lehr- und Forschungsspektrum der Medizinischen Fakultät sowie der gesamten Universität Rostock bereichert und für angrenzende Forschungsinstitute im Lande nutzbar ist. Die Verwertung der Forschungsergebnisse des Proteom-Zentrums Rostock erfolgt in Form von wissenschaftlichen Publikationen, Vorträgen, Postern sowie Teilnahme und Organisation von Podiumsdiskussionen, Kongressen und Messeauftritten. Darüber hinaus wurden Patente angemeldet, welche die Grundlage für Ausgründungen darstellen. Bereits stattgefundene Firmenausgründungen sind zu verzeichnen (Fa. IndyMed). Mit dem „Koichi Tanaka Mass Spectrometry Research Laboratory“ (Japan) wurde im September 2003 eine Vereinbarung zur engen Zusammenarbeit mit dem Proteom-Zentrum Rostock unterzeichnet. Das Proteom-Zentrum Rostock fungiert seit März 2003 als Referenzlabor für die Fa. Shimadzu. Die Aktivitäten des Proteom-Zentrums Rostock wurden breiten Kreisen der interessierten Bevölkerung in einer Vielzahl von Pressemitteilungen und Medienaktivitäten (TV-, Rundfunk-Beiträge sowie Podiumsdiskussionen) vorgestellt. Die Bundesministerin für Bildung und Forschung, Frau Bulmahn, sowie Vertreter der Landesregierung MecklenburgVorpommern statteten dem Proteom-Zentrum Rostock ihren Besuch ab.

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About the Proteome Center Rostock With the implementation of the Proteome Center Rostock in 1999 and the appointment of the first professorship for Proteome Research in Germany (Prof. Glocker), the State of Mecklenburg-Western Pommerania with support of the ministry of education and research, the ministry of economics, the University of Rostock, the City of Rostock and the „SteinbeisStiftung“ by terms of a "private-public partnership" has set the foundation for a sustaining research organisation that coins the scientific landscape, particularly the location of research in Rostock, in the field of proteome research / functional genomics and bioinformatics with close relations to clinical applications. Within the brief time of only a few years, a research center with international recognition has developed, as is exemplified by the attendance of Koichi Tanaka at the 2nd Proteome Forum in Rostock in September 2003, and is reflected by the high degree of national and international networking. The international conference "2nd Proteome Forum in Rostock" was part of the BMBF-program "Jahr der Chemie 2003". The University of Rostock has inaugurated the Department for Proteome Research that adds to the teaching and research competencies of the Medical Faculty and the entire university and which is accessible for adjacent research institutions in the State. Research results are published in scientific journals, as oral presentations, posters and by the participation in and organisation of scientific conferences, panel discussions, and by attending economic exhibitions. In addition, patents have been filed that shall function as the base of spin-off foundations, the first of them is IndyMED. In September 2003 the Proteome Center Rostock has signed a collaborative agreement with the "Koichi Tanaka Mass Spectrometry Research Laboratory" (Japan) and since March 2003 the Proteome Center Rostock has been functioning as reference laboratory for Shimadzu. The activities of the Proteome Center Rostock were made public to a broad audience by numerous press releases and contributions to media shows (TV-, broadcast and panel discussions). Mrs. Bulmahn, head of the German ministry of research and education, as well as representatives of the State government of Mecklenburg- Western Pommerania visited the Proteome Center Rostock on various occasions.

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Veranstaltungsstandorte und Tagungsräume / Locations and Conference Rooms

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Hörsaalgebäude Chemie / Chemistry lecture auditorium

Workshop 3

Internet Access

Poster (1-61)

Lunch seminar agilent

Garderobe

2OG

Raum 201

Poster Tagungsbüro

1OG August Michaelis Hörsaal

Hositrad Waters

Café 4

August Michaelis Hörsaal

Shimadzu

Vorbereitung 2

Bruker

Café 3

Science S.

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EG WC

Hörsaalgebäude Biologie / Biology lecture auditorium

2OG

Raum 201

Raum 202

Workshop 1

Workshop 2

Poster (62-122)

Lunch seminar Lunch seminar WATERS WATERS

GSG Thermo

Poster

Café 2

Proteome A.

1OG Hans Spemann Hörsaal

Karl von Frisch Hörsaal

Burle MasCom

Café 1

Hans Spemann Hörsaal

Vorbereitung 1

Agilent

Axel Semrau ABI Applied

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EG Karl von Frisch Hörsaal

WC

Danksagung / Acknowledgements Wir möchten uns bei den aufgeführten Firmen für ihre großzügige Unterstützung bedanken. We would like to acknowledge the generous contributions of the following companies.

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Tagungsprogramm – Übersicht / Program Overview Sunday, 6th of March, 2005 12:00 Registration Lecture auditorium Biology and Chemistry, Albert-Einstein-Str. 14:00 Workshops 1 - 3 Lecture auditorium Biology and Chemistry, Albert-Einstein-Str. 18:00 Conference Opening Aula, University main building, Universitätsplatz 18:30 Wolfgang - Paul Lecture Aula, University main building, Universitätsplatz 20:00 Welcome Reception Mayor´s hall City of Rostock, Neuer Markt Monday, 7th of March, 2005 (Lecture auditorium Biology and Chemistry) 08:00 Poster session P1 10:00 Plenary lecture HV1 10:45 Lectures V11-V15, V21-V25, V31-V35 12:25 Lunch / lunch seminars 13:30 Lectures V41-V45, V51-V55, V61-V65 16:10 Coffee break 17:00 Company presentations F1-F10 18:00 Plenary lecture HV2 Tuesday, 8th of March, 2005 (Lecture auditorium Biology and Chemistry) 08:00 Poster session P2 10:00 Plenary lecture HV3 10:45 Lectures V71-V75, V81-V84, V91-V95 12:25 Lunch / lunch seminar 13:30 Award celebration 15:30 Coffee break 16:30 General assembly 19:00 Conference dinner: "Teepott", Warnemünde, Strandpromenade Wednesday, 9th of March, 2005 (Lecture auditorium Biology and Chemistry) 09:00 Lectures V101-V104, V111-V114, V121-V125 10:40 Coffee break 11:40 Plenary lecture HV4 12:25 Poster awards / Closing remarks / Farewell

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Workshops 1) Workshop: FTICR-Mass Spectrometry Neue analytische Anwendungen der FTICRMassenspektrometrie

Leiter: Prof. Dr. Dr. h.c. Michael Przybylski

2) Workshop: off-line LC - MALDI Coupling off-line Kopplung cHPLC - MALDI-TOF. Neue Möglichkeiten und Applikationen.

Leiter: Prof. Dr. Michael Hecker

3) Workshop: ICP-MS Methods Neue Entwicklungen in der induktiv gekoppelten Plasmamassenspektrometrie (ICP-MS)

Leiter: Dr. habil. J. Sabine Becker, Dr. Jochen Vogl

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Hauptvortragende / Plenary lecturers Nico M. M. Nibbering, Free University, Amsterdam, NL

Chemistry and Mass Spectrometry (Wolfgang - Paul Vortrag) Frantisek Turecek, University of Washington, Seattle, USA

Biomolecular Radicals in the Gas Phase: Mass Spectrometry and Theory (HV 1) Peter Roepstorff, University of Southern Denmark, Odense, DK

Recent trends in analysis of large biomolecules by mass spectrometry (HV 2) H.-Jürgen Kluge, Gesellschaft für Schwerionenforschung mbH, Darmstadt, D

Präzisionsmassenmessungen an Radionukliden in Ionenfallen und Speicherringen (HV 3) Witold Danikiewicz, Polish Academy of Sciences, Warszawa, PL

The Use of Electrospray - Triple Quadrupole Mass Spectrometer as the Chemical Reactor: Studies on the Gas-Phase Chemistry of Aromatic Carbanions (HV 4)

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Tagungsprogramm / Scientific Program

Lectures / Workshops: Sunday, 6th of March, 2005 Time

Conf. part

Session / Chairman / Room

12.00 – 17.30

Registration

Registration / Poster setup Foyers, Lecture auditoriums Biology and Chemistry

14.00 – 17.30

Workshops WS1 WS2 WS3

WS 1: FTICR-Mass Spectrometry Chair: M. Przybylski Room 201, Biology

WS 2: off-line LC - MALDI Coupling Chair: M. Hecker Room 202, Biology

WS 3: ICP-MS Methods Chair: J. S. Becker, J. Vogl Room 201, Chemistry

18.00 – 18.30

Conference opening

Chair: J. Grotemeyer / M.O. Glocker Aula, University main building

18.30 – 20.00

Wolfgang Paul lecture

Nicco Nibbering Chemistry and Mass Spectrometry Chair: J. Grotemeyer Aula, University main building

20.00 – 22.00

Welcome reception

Reception Mayor´s hall, City of Rostock

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Posters: Monday, 7th of March, 2005 Time

Conf. part

Session / Chairman / Room

8.00 – 10.00

Poster session P1

Odd numbered posters Lecture auditoriums Biology and Chemistry

P1 XACT: Development of a New Method for Studying Protein-Protein Interactions Ahrends R ., Kirsch D., Spengler B., Friedhoff P. P3 Fast monitoring of the therapeutic effect of antibiotics in exhaled air by ion mobility spectrometry J.I. Baumbach, W. Vautz, V. Ruzsanyi, L. Freitag P5 Detection and quantification of microbial metabolites S. Bessenyei, D. Zimmermann, M. Hartmann, A. Grundmann, V. Ruzsanyi, W. Vautz, J. Nolte, J.I. Baumbach P7 Structural determination of neuronal Tau protein using Fourier Transform-ICRMS J. Su. Becker, A. Dumitrel, M. Przybylski P9 Capillary electrophoresis ICP-MS for separation of lanthanides in water samples from Israel C. Pickhardt, A.K. Malik, U. Seeling, A. Izmer, M. Zoriy, T. Platzner, I. Segal, N. Teplyakov, L. Halicz, J.S.Becker P11 Isotope ratio measurements of uranium on biological surface by LA-ICP-MS with a cooled laser ablation chamber in microscale range Myroslav V. Zoriy, M. Kayser, A. Izmer, C. Pickhardt, J.S. Becker P13 Low level detection of styrene oxide adducts to nucleotides using HPLC/ESI-MS Ulf Bergmann, Michael Linscheid P15 Towards Understanding the Fragmentation Mechanisms of Protonated Peptides: Competing Pathways for Val-Asn, Val-Gln and Val-Lys Christian Bleiholder, Béla Paizs, Sándor Suhai P17 Analysis of cellular protein translocation events using a combined approach of subcellular fractionation, 2D-Polyacrylamide Gel Electrophoresis and MALDITOF-MS Jadranka Milosevic, Patrick Bulau, Klaus T. Preissner, Günter Lochnit, Werner Seeger, Oliver Eickelberg P19 Mass Spectrometric Characterization of Protein Derivatives Modified with Amino and Thiol Selective Fluorescence Dyes for Use in Proteome Research H. Christoph, R. Woisch, R. Gohlke, M. O. Glocker, H.-J. Thiesen P21 Charakterisierung von Glycoproteinen mittels Massenspektrometrie: CE-ESITOF-MS and LC-ESI-IT-MSn U. Demelbauer, C. Neusüß, M. Pelzing, M. Macht, K. Schubert P23 Demands on HPLC in the Post-Genomics Era H.J. Wirth, P. Dawes, E. Dawes, B. Pfeffer 23

P25 Anwendung der ESI-FT-ICR-Massenspektrometrie zur Untersuchung des Startvorganges radikalischer Polymerisationen M. Buback, H. Frauendorf, F. Günzler, P. Vana P27 GC/MS analysis of disease markers the in breath of critically ill patients Fuchs Patricia, Miekisch Wolfram, Noeldge-Schomburg Gabriele, Schubert Jochen P29 Mass spectrometric characterization of peptides resulting from enzymatic digestion of recombinant human tropoelastin Melkamu Getie, Christian E.H. Schmelzer, Anthony S. Weiss, Reinhard H.H. Neubert P31 Fully Automated Liquid Injection Field Desorption/Ionization (LIFDI) Mass Spectrometry for High Throughput Screening of Compounds with Various Polarity Jens Griep-Raming, H. Bernhard Linden P33 Mapping nucleotide induced conformational changes in the Escherichia coli Hsp70 chaperone DnaK using amide hydrogen exchange and mass spectrometry Graf Cristian, Wolfgang Rist, Bernd Bukau und Matthias P. Mayer P35 Liquid Injection Field Desorption Ionisation am Flugzeit-Massenspektrometer (LIFDI-TOF-MS) Karl-Heinz Gunzelmann, Hans-Martin Hutmacher, Joachim Richert,Rainer Wolf P37 A quantitative proteomic strategy to study leaf senescence in Arabidopsis thaliana by metabolic labeling and mass spectrometry R. Hebeler, Kai Reidegeld, P. P. Dijkwel, Marcel J. G. Sturre, H. E. Meyer, B. Warscheid P39 Salt-free In-gel digestion of proteins in salt-free media by covalently modified trypsin. Henrik Thomas, Marek Sebela, Andrej Shevchenko P41 Analyse von Daten der bildgebenden MS durch visuelle Verfahren A. Hester, W. Bouschen, A. Leisner, K. Maaß, C. Paschke, B. Spengler P43 Thin-layer chromatography combined with diode laser desorption/atmospheric pressure chemical ionization mass spectrometry Song Peng, Norman Ahlmann, Kerstin Kunze, Walter Nigge, Michael Edler, Thorsten Hoffmann, Joachim Franzke P45 Determination of Iodine Species in the Coastal Atmosphere using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) H. Chen, R.Brandt, R.Bandur, N.Jakubowski, K.G.Heumann, T.Hoffmann P47 Proteome Analysis of Escherichia coli using High-Performance Liquid Chromatography and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Christian Ihling, Andrea Sinz P49 Differential proteome analysis of tonsil material from children with chronic tonsillitis in comparison with hyperplasia in order to identify diagnostic marker proteins Tino Just, Evariste Gafumbegete, Jan Gramberg, Ines Prüfer, Bruno Ringel, Hans Wilhelm Pau, Michael O. Glocker P51 Untersuchung der Solvolyse von tert.-Butylhalogeniden mittels ESI MS Björn Klein, Francesco Fabbretti, Jürgen O. Metzger 24

P53 Therapeutisches Drug Monitoring (TDM): Quantifizierung von antiretroviralen Pharmaka aus Plasma, Serum, Vollblut und Dried Blood Spots (DBS) mittels LC/MS/MS Therese Koal, Heike Burhenne, Klaus Resch, Michal Svoboda, Volkhard Kaever P55 Phosphohydroxyprolin ist eine proteinogene Aminosäure Axel Kühlberg, Mark Haid, Sabine Metzger P57 Kinetic Energy Release am Beispiel ungewöhnlicher Doppelprotonentransferreaktionen Alexandra E. Kulle, Stefan Schweiger, Guntram Rauhut, Jürgen Grotemeyer P59 Charakterisierung der Struktur von Molekülionen und Untersuchung von Ion– Molekül-Reaktionen durch Ionenmobilitätsspektrometrie R. Laudien, T. Beitz, B. Kallies, H.-G. Löhmannsröben P61 Multiplex Assay Monitoring by Means of ESI-MS for Proteolytic Activty Screening in Snake Venom André Liesener, Uwe Karst P63 Analysis of membrane proteins in mouse brain – The power of multi-dimensional separation Christiane Lohaus, Andreas Nolte, Helmut E. Meyer, Katrin Marcus P65 Structural characterization of Shiga toxin 1-binding glycosphingolipids in crude TLC overlay assay-derived silica gel extracts by nanoESI-QTOF mass spectrometry Iris Meisen, Alexander W. Friedrich, Helge Karch, Jasna Peter-Katalinic, Johannes Müthing P67 Vergleich verschiedener Enzyme auf Eignung für die massenspektrometrische Analyse von Membranproteinen Björn Meyer, Sandra Hofmann, Carsten Corvey, Khalid Siddiqui, Bernd Ludwig, Michael Karas P69 Mass spectrometric analyses of cyanobacterial proteomes: Studies of the salt stress response and the phosphoproteome of Synechocystis PCC 6803 Stefan Mikkat, Sabine Fulda, Martin Hagemann P71 Infrared Multiphoton Dissociation of peptides released by in-capillary proteolysis M. Mormann, G. Pohlentz, S. Kölbl, J. Peter-Katalinic P73 Higher sample information content by combing magnetic bead based fractionation and LC-MALDI-TOF/TOF on complex biological samples Dagmar Niemeyer, Sören-Oliver Deininger, Arndt Asperger, Marcus Macht P75 Epitope Elucidation of C-terminal Amyloid Precursor Protein (C-APP) Specific Antibodies of Alzheimer's Disease by High Resolution Mass Spectrometry Gabriela Paraschiv, Xiaodan Tian, Michael Przybylski P77 Bestimmung der Elementarzusammensetzung von Fullerenderivaten mittels hochauflösender API-oTOF-Technologie Matthias Pelzing, Nadine Bucci, Bianca Flock, Jürgen H. Gross, Thomas J. J. Müller P79 Vergleich zweier massenspektrometrischer Detektoren hinsichtlich ihrer Empfindlichkeit gegenüber Pestiziden und die Untersuchung von Fragmentierungsmustern Manuela Peschka, Thomas P. Knepper, 25

P81 Analyse eines wässrigen Extraktes von Früchten der Gattung Aronia mittels HPLC-MS und FTICR-MS Stefan Pieper, Michael W. Linscheid P83 MATI-Spektroskopie an Pyrimidin Mikko Riese, Jürgen Grotemeyer P85 New tools for nanoLC-FTICR-MS measurements Andreas Römpp, Ioana Taban, Mark Duursma, Vinh An Thieu, Dieter Kirsch, Bernhard Spengler, Ron Heeren P87 Thermostable trypsin derivates for enhanced in-gel digestion in high throughput proteomics Natalie Schmalz, Marek Šebela, Tatána Drhlíková, Henrik Thomas, Andrej Shevchenko P89 Rapid Drug Monitoring in Biological Matrices Using SPME // LC-MS/MS N. Scherzer, P. Fuchs, W. Miekisch, J. Schubert, H. Lord, J. Pawliszyn, B. Drewelow, R.G. Mundkowski P91 Studying Calmodulin-Adenylyl Cyclase VIII Interaction with Chemical CrossLinking and FTICR Mass Spectrometry Andreas Schmidt, Daniela M. Schulz, Stefan Kalkhof, Christian Ihling, Annette G. Beck-Sickinger, Dermot M.F. Cooper, Andrea Sinz P93 Massenspektrometrische Untersuchungen zur Bildung von Polymeren in atmosphärischen sekundären organischen Aerosolen Mathias Schott, Jörg Warnke, Thorsten Hoffmann P95 Purification and Characterization of the Tetrameric Complex Between Annexin II and p11 from Porcine Small Intestine Daniela M. Schulz, Andreas Schmidt, Olaf Zschörnig, Stefan Kalkhof, Christian Ihling, Andrea Sinz P97 Ethylenediamminedichloroplatinum and Cisplatin: A Study by Electrospray Ionization - High Resolution Mass Spectrometry A. Springer, M. Linscheid P99 A nano electrospray ionization - ion mobility spectrometer coupled to ion trap mass spectrometry for fast separation and structure elucidation of dendrimers and their by-products Ludwig Schwarz, Michael Linscheid P101 Mass spectrometric studies of the maleonitrile tetrathia crown ethers and their complexes with different transition metal ions I. Starke, S. Fürstenberg, H. Müller, H.-J. Holdt, E. Kleinpeter P103 SARAMIS A NEW WAY OF DATA MANAGEMENT OF MALDI-TOF MS FOR MICROORGANISM-IDENTIFICATION M. Erhard, W. Kallow, S. Sauermann P105 Identification of toxicologically relevant proteins from rat liver Kareen Tenz, Stephanie Lamer, Axel Oberemm, Michael Schümann, Eberhard Krause P107 Entwicklung eines quasi-kontinuierlichen Messverfahrens zur Überwachung von Vergasungsanlagen mit dem Ziel der Teerminderung Schulz-Tönnies, Karen, Neubauer, York, Behrendt, Frank 26

P109 LC-MS/MS and MS-BLAST for Automated Homology-Based Proteomics in Dunaliella salina Waridel P., Katz A., Pick U., Shevchenko A. P111 A novel strategy for the analysis of phosphopeptides, coupling N-terminal peptide derivatisation and HPLC separation with mass spectrometry M. Snel, I. Campuzano, E. Claude, T. McKenna, Marc Kipping, J. Langridge P113 A novel LC-MS based approach to qualitative and quantitative protein profiling and biomarker discovery Scott Geromanos, Keith Richardson, Phillip Young, Richard Denny, Kieran Neeson, Therese McKenna, Matthew Kennedy, Craig Doreschel, Marc Gorenstein, Guo-Zhong Li, Timothy Riley, Jeffrey C. Silva, James Langridge P115 Mass Spectrometric Proteome Analysis of Skeletal Muscle Tissue from Inbred Fat Mouse Strain in Comparison to Normal Weight Mouse Lines; On the Way to Detect Marker Proteins for Obesity M.R Wiechert, G.A Brockmann, U. Renne, M.O.Glocker P117 ESI-MS-Untersuchungen zur intrazellulären Phosphorylierung des Drosophila Transmembranproteins Crumbs Nicola Wiethölter, Susann Fornaçon, Elisabeth Knust, Sabine Metzger P119 Neue Strategien in der Strukturaufklärung durch LC/FT-MS Rainer Wolf, Michael Simon, P121 De Novo Sequencing using MALDI QIT TOF MSn – Application of a Novel Sequencing Algorithm to Clinical Proteome Samples H. Montgomery, C. Koy, M. May, J. Broughton, M. Resch, S. Iwamoto, M.O. Glocker, and K. Tanaka

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Lectures: Monday, 7th of March, 2005 Time

Conf. part

Session / Chairman / Room

10.00 – 10.45

Plenary lecture HV1

Frantisek Turecek Biomolecular Radicals in the Gas Phase: Mass Spectrometry and Theory Chair: K. Heumann Hans Spemann lecture hall, Biology

10.45 – 12.25

Lectures V01 V02 V03

12.25 – 13.30

Lunch seminars

V 01: Proteomics I: Post-translational modifications Chair: D. Suckau Hans Spemann lecture hall, Biology

V 02: Instrumentation I: New methods Chair: B. Lindner August Michaelis lecture hall, Chemistry

V 03: Environmental applications and basic research Chair: T. Knepper Karl von Frisch lecture hall, Biology

V11: J. Sabine Becker New horizons in brain research by LA-ICP-MS: From metallomics to imaging of thin section of brain tissues

V21: Nina Morgner Laserdesorption von Biomolekül-Ionen aus Mikrotröpfchen

V31: Anja These Die Kopplung von SEC und Q-TOF-MS offenbart den hochsystematischen Aufbau von Fulvinsäuren

V12: Martin Zeller Analysis of Posttranslationally Modified Peptides and Proteins by High Resolution ECD FTMS

V22: Marten Snel Parallel Post Source Decay MALDI-TOF – Principles and Application to Proteomics

V32: Marco Bernhard Monitoring and Fate of Polar Persistent Pollutans in Waste and Surface Water in Europe

V13: Clementine Klemm Mass spectrometric analysis of post-translational modifications of STAT1

V23: R. Woisch Cryodetektion mit MALDITOF-MS. Eine neue Methode für die Routineanalyse von großen Biomolekülen

V33: Claudia Martin Analyse von Galaxolid und Tonalid mittels ’TripleQuadrupole Linear Iontrap MS

V14: Tobias Langrock Analysis of hydroxylated prolyl and lysyl residues in collagens

V24: Henning Urlaub Validierung von Proteinidentifizierungen durch Multiple Reaction Monitoring (MRM)-getriggerte LC/MS/MS Acquisition

V34: A. Grundmann TD/GC/MS zur Atemluftanalyse

V15: Katrin Marcus Tryptic transpeptidation products observed in proteome analysis by LCMS/MS

V25: Aleš Svatoš Fast Prototyping of Hydrophobic Disposable Polymer Support Arrays for Matrix-assisted Laser Desorption/Ionization Time-ofFlight Mass Spectrometry of Proteins by Atmospheric Molding

V35: Ulrich Boesl Schneller simultaner Nachweis von Stickoxiden: Kombination moderner Methoden mit Methoden aus dem Wissensschatz der MSGrundlagenforschung

LS01: Waters Rooms 201 and 202, Biology Mensa / Restaurants

Lunch

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LS02: Agilent Room 201, Chemistry

Lectures: Monday, 7th of March, 2005 - continued Time

Conf. part

13.30 – 16.10

Lectures V04 V05 V06

16.10 – 17.00

Coffee break

Session / Chairman / Room

V04: Proteomics II: Glycoproteomics Chair: J. Peter-Katalinic August Michaelis lecture hall, Chemistry

V05: Instrumentation II: Hyphenated MS analysis Chair: J. Wesener Hans Spemann lecture hall, Biology

V06: Bioactive compounds and metabolites Chair: M. Svoboda Karl von Frisch lecture hall, Biology

V41: Martin Froesch High-Peformance Glycoscreening and Sequencing by chipenhanced ESI-FTICR Mass Spectrometry

V51: Martin Köstler Entwicklung einer rasternden Atmosphärendruck (AP)MALDI Quelle für das Finnigan LTQ-FT Massenspektrometer

V61: J.I. Baumbach Metabolites in Human Breath: Ion Mobility Spectrometers as Diagnostic Tools for Markers of Lung Diseases

V42: Matthias Pelzing Charakterisierung von komplexen Glykoproteinen mittels Kapillarelektrophorese- ESIoTOF am Beispiel von Erythropoietin (EPO)

V52: Klaus Dreisewerd Liquid-matrix-assisted infrared laser desorption/ionization orthogonal-time-of-flight mass spectrometry for the analysis of gangliosides directly from thinlayer chromatography plates

V62: D. Schwudke Unraveling Cellular Lipidomes by Simultaneous Multiple Neutral Loss Scanning and Multiple Precursor ion Scanning

V43: Koichi Tanaka Stabilization of Sialic Acid in MALDI-MS and Improvement of MS/MS Performance by Amidation

V53: Jens Grote An Interface for Coupling Surface Plasmon Resonance and Mass Spectrometry Experiments

V63: J. Lembcke Rapid quantification of free and esterified phytosterols in serum using APPI-LCMS/MS

V44: G. Pohlentz In-capillary proteolytic digest: Identification and/or Sequencing of hemoglobins directly from blood samples and structure elucidation of glycopeptides

V54: Friedrich Mandel CHIP-LCMS: A New Format and Interface for Nano-LCMS for Proteomics

V64: R. Zimmermann Time-of-Flight mass spectrometry as fast detector for two-dimensional comprehensive gas chromatography (GCxGCTOFMS): Characterization of highly complex samples in Metabolomics and environmental analysis

V45: B. Lindner Massenspektrometrische Analyse der temperaturabhängigen Variation und der speziesspezifischen Diversität des Lipopolysaccharides des Pesterregers Yersinia pestis

V55: Joerg Niebel NanoLC coupling to ESI Chip

V65: M. Möder Structure assignment of isomeric 4-nonylphenols by GC-MSn

Foyers, Lecture auditoriums Biology and Chemistry

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Lectures: Monday, 7th of March, 2005 - continued Time

Conf. part

17.00 – 18.00

Company lectures F1 F2

Session / Chairman / Room

F1: LC-MS / ESI-MS Chair: J.S. Becker August Michaelis lecture hall, Chemistry

F2: MS equipment and accessories Chair: K.-P. Wancek Hans Spemann lecture hall, Biology

F01: Linden: H. B. Linden Hyphenated Liquid Injection Field Desorption Ionization

F02: Sunyx: S. Goethel "MPep MALDI Chips: Eine neue Generation vorbeschichteter Targets zur LC-MALDI TOF Analytik"

F03: ABI: H. Sommer F04: Thermo: K. Scheffler Das API 5000TM LC/MS/MS System: Das erste Proteinidentifizierung mit der neuen vMALDIAPI Massenspektrometer mit QJet TM Ionenfalle von Thermo Electron Technologie für bisher unerreichte Sensitivität in der Quantifizierung kleiner Moleküle

18.00 – 18.45

Plenary lecture HV2

F05: Waters: J. Rontree Ultra Performance Liquid Chromatography and its impact on Mass Spectrometry

F06: Hositrad: D. Kimmel Research MS/MS Systems and Components from Extrel CMS

F07: Shimadzu: N. Loftus Trapping ions - a brief overview of an quadrupole ion trap time of flight mass analyser coupled with electrospray

F08: Agilent: W. Hepp Cerity ECM - Die elektronische Bibliothek für MS-Daten

F09: Bruker: U. Rapp Exakte Massenmessungen mit einem ESI-TOF - Ersatz der Sektorfeldgeräte?

F10: Science Serve: M. Weisser ACD/Labs Software für die Massenspektrometrie: Datenextraktion, Analyse, Strukturintegration und Vorhersage – alles in einer Umgebung

Peter Roepstorff Recent trends in analysis of large biomolecules by mass spectrometry Chair: M.O. Glocker August Michaelis lecture hall, Chemistry

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Posters: Tuesday, 8th of March, 2005 Time

Conf. part

Session / Chairman / Room

8.00 – 10.00

Poster session P2

Even numbered posters Lecture auditoriums Biology and Chemistry

P2 Ungewöhnliche Reaktionen von Indolizinen mit Chloroform in methanolischer Lösung unter Lichteinfluss M. Bartoszek, H. Sonnenschein P4 Statistical approaches for the analysis of lung diseases by means of ion mobility spectrometry J.I. Baumbach, S. Bader, W. Urfer P6 Tracer studies on human brain proteins by using MALDI-FTICR-MS and LAICP-MS after 2D gel electrophoresis J. Susanne Becker, Miroslav V. Zoriy, Carola Pickhardt, Michael Przybylski, J. Sabine Becker P8 Imaging of metals in human and rat brain tumors by laser ablation induction plasma coupled mass spectrometry C. Pickhardt, Markus Dehnhardt, M. V. Zoriy, Andreas Bauer, Andreas Matusch, Guido Reifenberger, Karl Zilles, J. Sabine Becker P10 Determination of Ra-226 in mineral waters at ultratrace level by ICP-MS M.V. Zoriy, Z. Varga, P. Ostapczuk, C. Pickhardt, R. Hille, J.S. Becker P12 Identification of novel proteins in DNA damage signalling pathways using 2DDIGE I. Bellahn, M. Szadkowski, P. Masterson, J. Prime, A. Moumen, S. Jackson, M. O’Connor, D. Carling P14 On-line reverse polarity capillary electrophoresis coupled to electrospray quadrupole time-of-flight mass spectrometry for analysis of glycopeptides from Schindler’s disease Laura Bindila, Jasna Peter-Katalinic, Alina Zamfir P16 Präparationsmethoden für hochauflösendes SMALDI Imaging W. Bouschen, O. Schulz, B. Spengler P18 Anwendung von Kryodetektoren in der Flugzeit-Massenspektrometrie P. Christ, F. Pröbst, S. Rutzinger, W. Seidel, S. Uchaikin P20 DETEKTION VON 6 SUDAN-FARBSTOFFEN, DIMETHYLGELB UND PARAROT IN GEWÜRZEN UND SOSSEN MIT HPLC/MS/MS Lutz Hartig, Katrin Hoenicke, Kristin von Czapiewski P22 Host-Bacteria-Interactions analyzed by 2D-PAGE and nanoLC-ESI-MS/MS W. Engst, C. Alpert, A. Gühler, M. Blaut P24 Investigation of Noncovalent Base Pairing of Nucleobase Functionalized bPeptides using ESI-FT-ICR-MS H. Frauendorf, N. Diezemann, P. Chakraborty, U. Diederichsen 33

P26 Simultaneous ESI/APCI Acquisition Using a Multimode Source in High Throughput Analysis Friedrich Mandel, Patrick D. Perkins, Steven M. Fischer, Douglas E. McIntyre, Wayne P. Duncan P28 Massenspektrometrische Untersuchung der Michael-Addition von Malonsäurediethylester an Acrylnitril: Direkter Nachweis von transienten Carbanionen Sven Fürmeier, Jürgen O. Metzger P30 A novel strategy for identification of protein-DNA contacts by MALDI-TOFMS/MS Hildegard Geyer, Rudolf Geyer, Vera Pingoud P32 REMPI-spektroskopische Untersuchungen an Dichlorbenzolen Angela Gaber, Zekeriya Altug, Jürgen Grotemeyer P34 Pigmentvergleich der rezenten Seelilie Hypalocrinus naresianus mit 240 Millionen Jahren alten Fossilien von Carnallicrinus carnalli Klaus Wolkenstein, Jürgen H. Gross, Matthias Pelzing, Uwe Demelbauer, Heinz F. Schöler P36 LC-MALDI on Disposable Prespotted AnchorChip Targets Martin Schuerenberg, Sven Brand, Christian Ziegmann, W. Jabs, Stephanie Hahner, D. Suckau P38 Cryodetector-mass spectrometric analysis of plasma proteins enables differentiation of HELLP patients from healthy women. J. Heitner, C. Koy, R. Woisch, M. Kreutzer, P. Serrano-Fernandez, R. Gohlke, T. Reimer, M.O. Glocker P40 Combinatorial Assembly of Heterometallic Double Helicates H. Herschbach, E. Leize, A. Van Dorsselaer, A. Marquis, J. M. Lehn P42 Wechselwirkung von UV-Strahlung mit der Haut – MALDI und ESRtomographische Messungen Hochkirch, U., Herrmann, W., Stößer, R., Borchert, H.H., Linscheid, M. P44 NanoLC MALDI MS und MS/MS – Methodenentwicklung für die Identifizierung von MHC Klasse I gebundenen tumor-assoziierten Peptidantigenen Sandra Hofmann, Matthias Glückmann, Sandra Kausche, Andrea Schmidt, Wolfgang Herr, Michael Karas P46 Untersuchung von RNA / DNA Dimeren und Minor Groove Binding Ligands mittels nano-ESI-MS Corina Hunger, Michael Karas P48 Untersuchungen zum Mechanismus der ISD-Fragmentierung am Beispiel der oxidierten Insulin B-Kette T. Jaskolla, M. Karas P50 Probing Laminin Self-Interaction by Chemical Cross-Linking and ESI-FTICR Mass Spectrometry Stefan Kalkhof, Sebastian Haehn, Christian Ihling, Neil Smyth, Andrea Sinz P52 ESI-MS Investigation of the Palladium-Catalyzed Allylic Substitution Reactions Larisa Knaack, Jürgen O. Metzger 34

P54 Characterization of Plasma Protein Pools from Rheumatoid Arthritis Patients Treated by Immune Adsorption Therapy – A Proteomic Approach Towards Understanding the Active Principle C. Koy, S. Drynda, B. Ringel, J. Kekow, H. Montgomery, K. Tanaka, M. O. Glocker P56 Development of a high-throughput procedure for the identification of proteinRNA cross-links from complex ribonucleoprotein particles (RNPs) based on IMAC and MALDI mass spectrometry Eva Kühn, Andrea Schmidt, Reinhard Lührmann, Henning Urlaub P58 Atmospheric Pressure Photoionization (APPI)-MS-MS coupled with membrane assisted extraction for determination of pharmaceutical and endocrine disrupting compounds H. Paschke, F. Lange, S. Schrader, M. Möder, W. Lorenz P60 Methionine Alkylation by Iodoacetamide May Interfere with Analysis of Peptide Serine/Threonine Phosphorylation by MS/MS Ralf Krüger, Chien-Wen Hung, Marina Edelson-Averbukh, Wolf D. Lehmann P62 Proteomics on Zebrafish Embryos Vinzenz Link, Carl-Philipp Heisenberg, Andrej Shevchenko P64 Flugzeiteffekte in der topologischen Massenspektrometrie Kai Maaß, Werner Bouschen, Arne Leisner, Bernhard Spengler P66 Mesoporöse TiO2-Membranen als MALDI-Probenträger zur Analyse von Phosphopeptiden in Peptidgemischen Anna Melikyan, Thomas Moritz, Michael W. Linscheid P68 SELDI-profiling and Identification of Differently Expressed Proteins in Body Fluids from Children Suffering from Juvenile Idiopathic Arthritis M. Meyer, R. Bogumil, K. Falk, B. Ringel, I. Prüfer, R. Nowack, J. Oppermann, H.-J. Thiesen P70 Identification of N-glycosylation sites and characterisation of N-glycan structure of soluble intercellular cell adhesion protein (Lec1 sICAM-1) by MALDI-FTICRMS A. Moise, E. Damoc, V. Otto, T. Schürpf, G. Folkers, M. Przybylski P72 Untersuchungen am Ablationsprozess der MALDI durch Postionisation Tassilo Muskat, Dirk Walbrodt, Jürgen Grotemeyer P74 Einsatz von LC/ESI-MS und GC/MS zur Bestimmung ausgewählter Pharmaka und ihrer Metaboliten J. Nolte, W. Nigge, M. Bataineh /, M. Grote P76 Monitoring nucleation reactions by coupling different reactors to an ESI Mass Spectrometer: about silicate species and zeolithe precursors Pelster S., Schüth F., Schrader W. P78 Analysis of synthetic chelating agents in surface and waste water by ion chromatography mass spectrometry Thomas P. Knepper, Andreas Werner, German Bogenschütz P80 Untersuchungen zum Auftreten von Barbituraten in deutschen Oberflächengewässern Manuela Peschka, Jan Eubeler, Thomas P. Knepper, 35

P82 LC/APCI-MS in Lipid Analysis Klaus Raith, Hany Farwanah, Christian Brenner, Christian Schmelzer, Reinhard Neubert P84 Mass Spectrometric Identification of Low Abundant Proteins from Human Plasma and Synovial Fluid after Depletion of Albumin by Free-FlowElectrophoresis B. Ringel, S. Drynda, S. Mikkat, C. Koy, I. Schinke, G.J. Eppert, H.-J. Thiesen, J. Kekow, M.O. Glocker P86 Kopplung eines Ionenmobilitätsspektrometers (IMS) an ein QuadrupolMassenspektrometer (Q-MS) mittels eines Interface zur Reduktion von Atmosphärendruck zum Hochvakuum Karsten Rowold, Mikko Riese, Uwe Eggers, Michael Karstens, Jürgen Grotemeyer P88 MALDI Imaging of Proteins and their Interaction Partners on Surfaces Christian E.H. Schmelzer, Andreas Huenerbein, Klaus Raith, Reinhard H.H. Neubert P90 Large-scale quantitative proteome analysis of apoptotic human cells using ICPL (Isotope-Coded Protein Label) and µLC-MALDI Alexander Schmidt, Cornelia Ciosto, Josef Kellermann, Friedrich Lottspeich P92 Multidimensional HPLC In Proteomics Oliver Schmidt, Thomas Schulenborg, Christiane Lohaus, Helmut E. Meyer, Katrin Marcus P94 Protein and Phosphoprotein Analysis Using a 4000 Q Trap mass spectrometer Thomas Schulenborg, David Fischer, Helmut E. Meyer, Fred van Leeuwen, Katrin Marcus P96 Enrichment of glycoproteins and glycopeptides supported by magnetic particles and detected by MALDI-TOF-MS Katrin Sparbier, Sonja Koch, Irina Keßler, Thomas Wenzel, Markus Kostrzewa P98 Biogenic iodine compounds in environmental samples and their determination by GC-MS Nicola Springer, Thorsten Hoffmann P100 Photoactivities of coated TiO2 in aqueous and water-oil system measured by HPLC-Ion trap mass spectrometry Guangzu Zhang, Michael Linscheid P102 LC-MS/MS in der Rückstandskontrolle – Kokzidiostatika in Eiern Michael Stephan, N. Palinsky, M. Ludewig P104 Gas-phase chemistry of 1,2,3,4-tetrahydrobenzopyran-2,3-dicarboxylic acid derivatives upon electron ionization by means of metastable mass spectrometry R. Szmigielski, W. Danikiewicz, K. Dolatowska, K. Wojciechowski P106 Pyridinium based ionic liquid matrices for the analysis of peptides by MALDIToF mass spectrometry Masoud Zabet-Moghaddam, Elmar Heinzle, Andreas Tholey P108 Aufreinigung und Analyse von Phosphopeptiden und His-Tag-Proteinen mittels eines funktionalisierten MALDI-Probentellers Tri Hoang, Ali Tinazli, Robert Tampé, Michael Karas

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P110 Parallel PSD analysis of 2D-gel spots from E.coli M. Snel, E. Claude, D. Kenny, T. McKenna, Marc Kipping, J. Langridge P112 NanoLC/MS In Quantitative and Qualitative Proteomics Jeff. W. Finch, Hong Ji Liu, Jim Langridge, Geoff Gerhardt, Matthew Kennedy, Steve. A. Cohen, K. Fadgen P114 High Performance Proteasome Proteomics using UV- and IR-MALDI FT-ICR Mass Spectrometry Reinhold Weber, Nikolay Youhnovski, Regina Preywisch, Marcus Groettrup, Michael Przybylski P116 A novel approach to stable isotope protein labeling for quantitative proteomics Sebastian Wiese, Kai Reidegeld, Bettina Warscheid P118 Capillary Electrophoresis-ESI-FTMS: Accurate Mass Measurement of Amino Acids in Lysated Bacterial Cells Matthias Witt, Christian Neusüss, Edward Baidoo P120 Determination of ganglioside expression in human gliosarcoma by chip electrospray tandem mass spectrometry Alina D. Zamfir, Laura Bindila, Željka Vukelic, Jasna Peter-Katalinic P122 Synthesis and structural characterization of polyubiquitin conjugates using high resolution mass spectrometry Ji Eun Jung, Marilena Manea, Martin Scheffner, Michael Przybylski

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Lectures: Tuesday, 8th of March, 2005 Time 10.00 – 10.45

10.45 – 12.25

Conf. part Plenary lecture HV3 Lectures V07 V08 V09

Session / Chairman / Room H.-Jürgen Kluge Präzisionsmassenmessungen an Radionukliden in Ionenfallen und Speicherringen Chair: L. Schweikhard Hans Spemann lecture hall, Biology V 07: Proteomics III: Biological applications Chair: M. Kussmann August Michaelis lecture hall, Chemistry V71: M. Kussmann Nutriproteomics: Health Promotion and Disease Prevention

V 08: Peptide and protein analysis I: amino acids and peptides Chair: M. Resch Karl von Frisch lecture hall, Biology

V72: K. Falk Proteome Analysis of Body Fluids from Children Suffering from Juvenile Idiopathic Arthritis and Comparison to Rheumatoid Arthritis from Adults Shows Differences in Calprotectin Expression

V82: Juri Rappsilber Peptide identification using peptide fragment ion vectors

V92: S. Dresen Multi Target Screening Analyse mit QTrap™ LCMS/MS und Bibliotheksuche zum Nachweis von 300 forensisch relevanten Drogen in Körperflüssigkeiten

V73: Sven Baumann A standardized approach of proteome profiling of human serum using magnetic bead separation and matrix assisted laser desorption/ionizationtime of flight mass spectrometry

V83: Bernhard Spengler Kompositionsanalyse und de Novo-Sequenzierung von Peptiden

V93: Stefan W. Toennes Bedeutung der Flugzeitmassenspektrometrie als HPLC-Detektor bei forensisch-toxikologischen Untersuchungen

V75: Jürgen Schäfer Novel MS based Strategies for Differential Quantitative Analysis of Complex Proteomes Lunch seminar Lunch

Chair: W. Weinmann S.W. Toennes Hans Spemann lecture hall, Biology

V81: Zekeriya Altug V91: Axel Besa Untersuchungen der Neue Möglichkeiten zur konformeren Anordnungen von Erhöhung der Tryptophan und der Empfindlichkeit: Das API Lebensdauern der zugehörigen 5000TM LC/MS/MS Systems angeregten elektronischen in der Quantifizierung kleiner Zustände mit Hilfe der REMPIMoleküle TOF-MS.

V74: Markus Schirle V84: S.Y. Vakhrushev Scoring proteomes by Combined strategy for analysis proteotypic peptides and mass of complex glycoconjugate spectrometry mixtures from urine of CDG patients by HPAEC-PAD and mass spectrometry with computational analysis

12.25 – 13.30

V 09: Forensic applications

V94: D. Kirsch Untersuchungen zum Alterungsverhalten von Kugelschreibereinträgen

V95: Matthias Frank Real-time analysis of individual bioaerosol particles and single cells with bioaerosol mass spectrometry LS03: Waters Rooms 201 and 202, Biology Mensa / Restaurants

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Lectures: Tuesday, 8th of March, 2005 - continued Time

Conf. part

Session / Chairman / Room

13.30 – 15.30

Award ceremony

Life Science Prize / Mattauch Herzog Prize / Wolfgang-Paul Prize

15.30 – 16.30

Coffee break

Foyers, Lecture auditoriums Biology and Chemistry

16.30 – 17.30

Society

General meeting

Chair: J. Grotemeyer August Michaelis lecture hall, Chemistry

Chair: J. Grotemeyer Hans Spemann lecture hall, Biology 19.00 – 22.00

Society

Conference Dinner Teepott, Warnemünde, Strandpromenade

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Lectures: Wednesday, 9th of March, 2005 Time

Conf. part

9.00 – 10.40

Lectures V10 V11 V12

Session / Chairman / Room V 10: Proteomics IV: Phosphoproteomics

V 12: MS in chemistry and natural products

Chair: M. Przybylski Hans Spemann lecture hall, Biology

V 11: Peptide and protein analysis II: protein interactions Chair: A. Sinz August Michaelis lecture hall, Chemistry

V101: Hanno Steen Stable Isotope-free Relative and Absolute Quantitation of Protein Phosphorylation Stoichiometry by Mass Spectrometry

V111: Andrea Sinz Mapping Protein Interfaces by Affinity Cross-Linking Combined with MALDI-TOF and ESI-FTICR Mass Spectrometry

V121: Karsten Levsen Die Fragmentierung von Even Electron Ionen

V102: C. Sykora Anreicherung und massenspektrometrische Identifizierung phosphorylierter Peptide

Chair: U. Boesl Karl von Frisch lecture hall, Biology

V112: Olaf Jahn V122: Daniel Kühne Characterization of peptideThe Proton Affinity of Liquid protein and protein-protein Crystal Model Compounds interactions using photoaffinity labeling and mass spectrometry

V103: Andreas Schlosser Phosphorylation site mapping by a multi-protease approach with highly specific phosphopeptide enrichment

V113: Jörg Oliver Thumfart Potential of functional proteomics: An epilepsy gene product slows K+ currents by assembly with Kv1.1polyprotein complexes

V123: S. Pelster Monitoring nucleation reactions by coupling different reactors to an ESI Mass Spectrometer: about silicate species and zeolithe precursors

V104: Ralf Krüger Evaluierung von LC-ICP-MS und LA-ICP-MS zur Bestimmung des Protein- oder ProteomPhosphorylierungsgrades

V114: G. Franke Quantiative Analysis of the Phosphotransferase System in Escherichia Coli by Metabolic Labeling and MALDI Mass Spectromery

V124: L. S. Santos Probing the Mechanism of Classical and the Supramolecular Co-Catalyzed Baylis-Hillman Reactions via ESI(+)-MS and MS/MS V125: Jürgen Schmidt Mit Tyramin auf Spurensuche im Schlafmohn

10.40 – 11.40

Coffee break

Foyers, Lecture auditoriums Biology and Chemistry

11.40 – 12.25

Plenary lecture HV4

Witold Danikiewicz The Use of Electrospray - Triple Quadrupole Mass Spectrometer as the Chemical Reactor: Studies on the Gas-Phase Chemistry of Aromatic Carbanions Chair: D. Kuck August Michaelis lecture hall, Chemistry

12.25 – 13.00

Society

Poster awards / Closing remarks / Farewell Chair: J. Grotemeyer / M.O. Glocker August Michaelis lecture hall, Chemistry

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Werbeseite 11 (Hositrad)

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Abstracts

Wolfgang-Paul-Vortrag / Wolfgang- Paul lecture

Mass Spectrometry and Chemistry Nico M.M. Nibbering Laser Centre and Chemistry Department Vrije Universiteit, De Boelelaan 1083 1081 HV Amsterdam, The Netherlands

Mass spectrometry has played and is still playing an important role in chemistry, although its focus has been shifted increasingly and successfully to biochemistry, biology and medicine in the past decade. Many developments in mass spectrometry have taken place during the career of the author over the last forty years. In the lecture selected examples of research, performed in his group during that period, will be presented including the use of a home-built Fourier transform ion cyclotron resonance mass spectrometer, field desorption ionization and field ionization kinetics (time-resolved mass spectrometry).

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Plenary lecture HV1 Biomolecular Radicals in the Gas Phase: Mass Spectrometry and Theory František Tureček Department of Chemistry, Bagley Hall, University of Washington, Seattle, WA 981951700, USA Many chemical and biochemical processes occur under conditions where the chemistry of the system is difficult to control and study in situ, e.g., in atmospheric reactions, high-energy reactions, combustion, radiation damage, etc. For such difficult to study systems, mass spectrometry offers three principal advantages: (1) the inert environment of the rarefied gas phase, (2) ion chemistry methods of generating the species of interest, and (3) efficient qualitative and quantitative analysis of reaction products, both ionic and neutral. This lecture will be concerned with mass spectrometric and computational studies of transient radicals that are formed in the complex process of radiation damage of nucleic acids. In particular, DNA radiation damage has been studied extensively for nucleic bases, nucleosides, and oligonucleotides in solution, crystal, and frozen glasses and a number of ingenious chemical and spectroscopic methods have been developed and applied to identify reactive intermediates [1]. In spite of many successes, there is still much to be learned about the early chemical steps of radiation damage where critical information is often missing or the data has been contradictory. An important type of radiation damage is triggered by low-energy electron attachment to the nucleobase to form an anion radical that is subsequently protonated by the solvent to form a radical which is formally a hydrogen atom adduct. A different process starts with solvent (water) radiolysis forming free hydrogen atoms that can exothermically attack the nucleobase to produce transient hydrogen atom adducts. The relative importance of these processes, the H-atom adduct structures, and their stability/reactivity are mostly unknown. In mass spectrometry, one can reverse the electron capture/protonation sequence by adding the proton first to produce a gas-phase cation. The gas-phase cations of welldefined structure are then discharged by femtosecond collisional electron transfer from a suitable atomic or molecular donor under conditions that preserve the structure of the incipient radical [2]. Mass analysis of radicals and their dissociation products is achieved after non-selective collisional ionization that provides more or less complete product analysis of the radical reactions. When combined with kinetic measurements, photodissociation, stable isotope labeling, and computational analysis of the potential energy surface, the mass spectrometric analysis furnishes a wealth of unique data to characterize the properties of transient intermediates. The systems that will be presented and discussed in the lecture include radicals of the nucleobases uracil, cytosine, and adenine, 2-deoxyribose derivatives, and phosphate radicals. It will be shown that these gas-phase radicals, cations, and anions have interesting and sometimes puzzling chemistry that can be explained qualitatively and, in favorable cases, even quantitatively by combining experiment and theory [3]. [1] S. Steenken: Chem. Rev. 1989, 879, 503-520. [2] F. Tureček: Top. Curr. Chem. 2003, 225, 77-125. [3] S. Vivekananda, M. Sadílek, X. Chen, L. E. Adams, and F. Tureček: J. Am. Soc. Mass Spectrom. 2004, 15, 1068-1079.

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Plenary lecture HV2 Recent trends in analysis of large biomolecules by mass spectrometry Peter Roepstorff Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK 5230 Odense M, Denmark New ionization methods developed between 1980 and 1990 have entirely changed the scope of mass spectrometry (MS) for analysis of large biomolecules. The presently dominating ionization modes are matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) combined with a variety of mass analyzers or combinations of analyzers of which especially the Time-of -Flight (TOF) and the Fourier Transform Ion Cyclotron Resonance (FT-ICR) analyzers in the past decade have been developed to a very high performance level. Analysis of large biomolecules can be performed by two approaches: Top-down based on analysis of the intact biomolecule and on subsequent fragmentation of this in the mass spectrometer, and bottom-up based on biochemical and chemical cleavage of the molecule followed by analysis of the now much smaller cleavage products by MS. Often a combination of the two approaches can be advantageous for detailed characterization of the biomolecules. The major application of MS of large biomolecules in biology is in protein studies and especially in the new field termed proteomics. Proteomics is mainly based on the bottom-up concept that takes advantage of the ability of contemporary MS techniques to analyze underivatized peptides at a sensitivity level comparable to the needs in cell biology and to analyze complex peptide mixtures. Proteomics strategies for protein identification relative to database information, assignment of protein modifications and identification of protein interaction partners will be described as well as examples of characterization of individual proteins by a top-down or a combined top-down bottom-up approach. The main focus of nucleic acid MS has been assignment of SNP’s for fine characterization of genomic variations and for assignment of nucleic acid modifications especially in RNA’s. Due to the large size of the nucleic acid molecules most nucleic acid MS is based on a bottom-up approach. The use of MS for analysis of carbohydrates, which represent the third major group of large group of biomolecules, is still limited. However, in combination with proteomics and protein characterization major progress has been made in characterizing the glycans in glycoproteins. In addition a number of applications of MS to characterize large carbohydrates, e.g. pectins, arabinoxylans and heparins, and the action of different enzymes on these has recently been published. These latter are all based on a bottom-up strategy. Overall mass spectrometry is now an essential tool for analysis of large biomolecules in natural and medical sciences and analysis of proteins, nucleic acids and carbohydrates by MS is now routine in many biological laboratories in academia as well as in industry.

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Plenary lecture HV3 Präzisionsmassenmessungen an Radionukliden in Ionenfallen und Speicherringen H.-Jürgen Kluge Gesellschaft für Schwerionenforschung mbH (GSI), Darmstadt and Universität Heidelberg Mass spectrometry is a very well established technique in many disciplines of pure and applied science. In nuclear physics, high-precision mass determinations are important to directly observe nuclear structure effects such as shell closures, pairing, onset of deformation or the limits of nuclear binding. In nuclear astrophysics, the mass of radionuclides is a crucial parameter for reliable calculations of nucleosynthesis processes. Furthermore, highly precise measurements of beta-decay energies are mandatory for nuclear-physics tests of the Standard Model as, for example, the verification of the conserved-vector-current hypothesis or the check of the unitarity of the quark mixing Cabibbo-Kobayashi-Maskawa matrix. In the last decade, new ideas have been realized for high-precision mass measurements of short-lived radionuclides which both use the principle of trapping and cooling. These were pioneered on the small scale of ion traps by setting up the triple-trap mass spectrometer ISOLTRAP at ISOLDE/CERN and on the large scale of storage rings by developing the Schottky and isochronous mass spectrometry for the experimental storage ring ESR at GSI/Darmstadt. In the mean time, a large fraction of all known masses in the chart of nuclei have been determined by both devices, and throughout the world many other Penning trap facilities at accelerators are operational, in the building-up stage, or planned.

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Plenary lecture HV4 The Use of Electrospray – Triple Quadrupole Mass Spectrometer as the Chemical Reactor: Studies on the Gas-Phase Chemistry of Aromatic Carbanions Witold Danikiewicz Institute of Organic Chemistry, Polish Academy of Sciences, ul. Kasprzaka 44/52, 01-224 Warszawa, Poland A triple-quadrupole mass spectrometer, equipped with an electrospray (ESI) ion source, is not only a powerful analytical tool but also a very efficient chemical reactor which can be used for studying various types of ion-molecule reactions in the gas phase. Ions formed in the atmospheric pressure region of the ESI ion source (see schematic diagram above, which shows API 365 mass spectrometer) are transferred to the mediumCurtain gas (with reagent vapors if necessary)

Medium pressure reaction zone

Q0

Collision gas (with reagent vapors if necessary)

Q1

Q2

Q3

Capillary Atmospheric pressure

~2 Torr

Collision cell

pressure zone of the source in which they undergo fragmentation and subsequent reactions with gaseous reagent delivered with the curtain gas. Selected product ions can be fragmented by collisions with the neutral gas in a collision cell or can undergo further ion+ R-H molecule reactions with the gaseous proton exchange reagents added to the collision gas. COO + O2 In the following presentation we oxidation would like to present the use of API 365 - CO + CO mass spectrometer for studying the 2 CID carboxylation formation and reactions of gas-phase X X phenide anions. These anions are formed benzoate phenide + Z-C-H anion anion further in the medium-pressure zone of the ion reactions formation of adducts (when X = NO , Z = source by decarboxylation of the electron withdrawing group) substituted benzoate anions generated by ESI.[1] The resulting phenide anions can react with a variety of gases as well as the vapors of liquid reagents. The reaction can take place either in a collision cell or in the mediumpressure zone of the ion source. Especially interesting are the reactions of mono-, di- and trinitrophenide anions (containing also some other substituents) with C-H acids of the appropriate acidity.[2] In these reaction anionic -adducts are formed which can undergo further transformations like aromatic nucleophilic substitution (SNAr) or substitution of hydrogen reactions. 2

2

Literature [1] T. Bieńkowski, W. Danikiewicz Rapid Commun. Mass Spectrom. 2003, 17, 697-705. [2] J. Am. Soc. Mass Spectrom. 2004, 15, 927-933.

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Lecture V11 New horizons in brain research by LA-ICP-MS: From metallomics to imaging of thin section of brain tissues J. Sabine Becker1, Miroslav Zoriy1, J. Susanne Becker2, Carola Pickhardt1, Nicola Palomero-Gallagher3, Markus Dehnhardt3, Andreas Matusch3, Michael Przybylski2 und Karl Zilles3 1

Central Division of Analytical Chemistry, Research Center Juelich, Germany. 2Laboratory of Analytical Chemistry, Department of Chemistry, University of Konstanz, Germany. 2 Institute of Medicine, Research Centre Juelich, D-52425 Juelich, Germany

For identification of metal-containing proteins and determination of Cu, Fe, Zn concentration in human brain proteins a combination of atomic and molecular mass spectrometric methods was applied. The protein mixtures were separated by two dimensional (2D) gel electrophoresis and the protein spots were fast screened by microlocal analysis using LA-ICP-MS (laser ablation inductively coupled plasma mass spectrometry) in respect to several elements, such as P, S, Cu, Zn and Fe. Selected protein spots in 2D gel containing these elements were identified after tryptic digestion by matrix assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Results of structure analysis of proteins by MALDI-FTICR-MS were combined with those of the direct element determination in protein spots with LA-ICPMS in order to characterize proteins in respect to their structure, sequence, phosphorylation state and metal content as well [1]. In order to study the formation of proteins containing Cu, Zn and Fe in a human brain sample isotopic-enriched tracers (54Fe, 65Cu and 67Zn) were doped to two-dimensional gels of separated Alzheimer-diseased brain proteins after 2D gel electrophoresis and analyzed by LA-ICP-MS and MALDI-FTICR-MS. Furthermore, LA-ICP-MS was used to produce images of element distribution of 20 µm thin sections of human brain tissues from the hippocampus. The quantitative determination of copper, zinc, uranium and thorium distribution in thin slices of human brain samples from the hippocampus was performed using matrix-matched laboratory standards. In addition, a new solution based calibration procedure using micronebulizer which was inserted directly in the laser ablation chamber was applied for validation of analytical data. The mass spectrometric analysis yielded an inhomogeneous distribution (layered structure) for P, S, Cu and Zn in thin brain sections of hippocampus [2]. The unique analytical capability and the limits of LA-ICP-MS will be demonstrated for the imaging of element distribution in thin cross sections of brain tissue from the hippocampus. In contrast, Th and U are more homogeneously distributed at a low concentration level with detection limits in the low ng g-1 range. Furthermore, the quantitative distribution of metals in thin slices of rat brain tumors is measured by LA-ICP-MS and compared to autoradiographs. In our studies, we found a very distinctive distribution of metal ions LA-ICP-MS provides new information on the spatial element distribution of the layered structure in thin sections of brain tissues. References [1] J. S. Becker, M. Zoriy, J. Su. Becker, C. Pickhardt, M. Przybylsky, J. Anal. At. Spectrom. 19 (2004) 149. [2] J. S. Becker, M. Zoriy, C. Pickhardt, N. Palomero-Gallagher, K. Zilles, Anal. Chem. 2005 (in press) .

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Lecture V12 Analysis of Posttranslationally Modified Peptides and Proteins by High Resolution ECD FTMS Martin Zeller, Wolfgang Metelmann-Strupat, Thomas Möhring, Kerstin Strupat and Helmut Münster Thermo Electron (Bremen) GmbH, Hanna-Kunath-Str. 11, 28199 Bremen, Germany During the last few years, electron capture dissociation (ECD) has rapidly emerged as an important alternative activation method especially for peptide and protein sequencing in Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS). The most commonly used activation methods such as collision induced dissociation (CID) or infrared multiphoton dissociation (IRMPD) induce dissociation by vibrational excitation of the parent ions, resulting in the cleavage of the weakest bonds. Within peptides, the backbone amide bond has the lowest energy barrier to dissociation and predominantly band y-ions are formed. However, substituents added in co- and posttranslational modifications (PTM) often dissociate with even lower energy barriers than those of backbone cleavage. This can result in more complex tandem mass spectra and eventually in the loss of the information of the attachment site of these substituents. In contrast, ECD induces a radical driven fragmentation and preserves PTM’s. We have used ECD for the analysis of various peptides and proteins to show that sites of modifications can be unambiguously determined. We applied ECD in different proteomic approaches, including data-dependent LC-ECD-FTMS and top-down characterization of intact proteins. The multiply charged ions, generated by nano electrospray ionization, were analyzed using a hybrid linear ion trap Fourier-transform mass spectrometer. Precursor ions were selected using the linear ion trap, and isolated ions were either fragmented by CID and passed to the FTICR analyzer for analysis, or passed directly to the ICR cell for activation by ECD before mass analysis. The resulting mass spectra are deconvoluted and compared to simulated spectra of these proteins to demonstrate determination of the PTM sites as well as accuracy of mass and isotopic pattern to show the potential of these approaches in proteomic experiments.

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Lecture V13 Mass spectrometric analysis of post-translational modifications of STAT1 Clementine Klemm, Torsten Meissner, Uwe Vinkemeier and Eberhard Krause Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125 Berlin, Germany STATs (signal transducers and activators of transcription) are highly conserved proteins which are involved in signal transduction and activation of transcription of target genes. The proteins convert extra cellular stimuli into a wide range of cellular processes. In response to interferon gamma STAT1 becomes phosphorylated at Tyr701 and dimerizes which leads to accumulation in the nucleus. An additional phosphorylation of a specific serine within the transactivation domain is necessary for full transcriptional activity. Recently a model has been proposed which suggested that methylation of Arg31 is required for DNA binding of STAT1, thus enhancing the cell growth-restraining activity of interferons. The present work describes the mass spectrometric determination of phosphorylation and methylation sites of the 87 kDa protein human STAT1. After the tryptic in-gel digestion of STAT1 the direct detection of phosphorylated peptides in the fingerprint spectrum failed due to signal suppression. Therefore a recently described modification strategy was applied which provides increased signal intensities for phosphorylated peptides in MALDI MS [1]. This was achieved by the ß-elimination of the phosphate group and the following addition of phenylethanethiol (PET). This reaction was performed in-gel prior to the tryptic digestion of STAT1. The results showed that the PET-derivatization of STAT1 allowed direct detection of the peptide sequences covering the phosphoserine position 727 which is normally suppressed in complex mixtures. Noteworthy, the PET-modified serine side-chain was stable under collision-induced dissociation conditions, thus enabling a reliable determination of the modification site by MALDI-TOF-TOF. In order to study the Arg31 methylation strep-tagged STAT1 was purified from stable transfected U3A cells and cleaved by AspN. Both nanoLC-ESI-MS and MALDI-MS revelaled that mass peaks which would match with Arg31-methylated sequences were definitely not present, whereas signals corresponding to the unmodified fragments 29-38 and 29-41 were found with considerable intensities. To exclude the possiblity that methylated peptides went undetected due to insufficient ionization, the ionization behavior of methylated STAT fragments was studied using equimolar mixtures of synthetic peptides. Since MALDI and ESI measurements yielded comparable signal intensities irrespective of methylation we concluded that STAT1 is not methylated at Arg31 to a significant extent. In summary, based on mass spectrometric analysis we provided evidence contradicting recent results about the role of Arg31 methylation in DNA binding of STAT1. In addition, we found that the methyltransferase inhibitor methylthioadenosine (MTA) inhibited also the phosphorylation of STAT1 [2]. Thus, alternative explanations to methylation have to be explored to understand the molecular mechanism of reduced interferon sensitivity of tumor cells which accumulate high levels of MTA. References [1] C. Klemm, S. Schröder, M. Glückmann, M. Beyermann, E. Krause (2004) Rapid Commun. Mass Spectrom. 18: 2697-2705 [2] T. Meissner, E. Krause, I. Lödige, U. Vinkemeier (2004) Cell 119: 587-590

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Lecture V14 Analysis of hydroxylated prolyl and lysyl residues in collagens Tobias Langrock , Natividad García-Villar , Ralf Hoffmann Bioanalytics, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, University of Leipzig

Collagens are the most abundant proteins in connective tissue, cartilage and bones. They can be divided into several subgroups, based on the exon structure of their genes and their functions, respectively[1]. All collagens are characterised by their unique triple helical structure, formed by three collagen chains, which display a high amount of glycine and proline. Hydroxylation of specific prolyl and lysyl residues is necessary for the stability of the triple helix. Even though degeneration of collagens is known to cause diseases like osteoporosis, the distribution of hydroxylysine (hyl) and the different hydroxyproline (hyp) isomers in collagens on a molecular level is rarely investigated. The aim of this study is to analyse the hyl and hyp content of various collagens. Especially the distribution of the four possible hyp isomers, cis-3-, cis-4-, trans-3- and trans-4hydroxyproline exhibits an analytical challenge, as these four compounds are all isobaric with leucine and isoleucine. Therefore at least these six amino acids (AAs) must be separated by liquid chromatography to allow their identification with MS. Here, we describe two chromatographic methods for the separation of most proteinogenic and hydroxylated AAs. A direct analysis can be applied using hydrophilic interaction chromatography (HILIC)[2], while separation on a C-18 phase requires pre-column derivatization, in our case performed with an analogue of marfey’s reagent, i.e., N2-(5fluoro-2,4-dinitrophenyl)-L-valine amide (L-FDVA) [3]. In both cases, MS-detection is essential for the identification of coeluting compounds. Literatur [1] T. Kreis and R. Vale: Guidebook to the Extracellular Matrix, Anchor, and Adhesion Proteins. A Sambrook & Tooze Publication at Oxford University Press, Secon d Edition, 380-408 [1999] [2] Bing-Yan Zhu, Colin T. Mant, Robert S. Hodges: Hydrophilic-interaction chromatography of peptides on hydrophilic and strong cation-exchange columns. Journal of Chromatography, 548, 13-24 [1991] [3] H. Brückner, C Keller-Hoehl: HPLC separation of DL-amino acids derivatized with N2-(5-fluoro-2,4-dinitrophenyl)-L-amino acid amids. Chromatographia, 30, 621-629 [1990]

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Lecture V15 Novel MS based Strategies for Differential Quantitative Analysis of Complex Proteomes Jürgen Schäfer, Christian Baumann, Karsten Kuhn, Stefan Kienle, Christian Hamon, Sandra Steiner, Josef Schwarz Proteome Sciences R&D GmbH&CoKG, Altenhöferallee 3, 60438 Frankfurt am Main The growing demand for novel and tailored therapeutic targets and reliable biomarkers for the diagnosis, prognosis and monitoring of diseases continues to stimulate innovations in "omics" technologies. In the past decade, new insights into biochemical pathways disturbed by pathologies could be gained by differential profiling of proteins using approaches such as 2D-gelelectrophoresis [1] combined with mass spectrometry. A number of candidate targets and biomarkers have already been delivered to the drug discovery pipelines [2]. Intensive efforts have been made to develop alternative technologies for quantitative differential gelindependent proteome analysis. These techniques include promising concepts involving stable-isotope labeling combined with mass spectrometry. We have developed a gelindependent peptide/protein tags for differential quantitative proteome analysis including the quantitative Protein Sequence Tag® (qPST™) and the Tandem Mass Tag® (TMT®) technology. The qPST™ is a further development of our previously published PST® procedure [3]. The method is based on N-terminal labelling of cyanogen bromide cleaved proteins using an unique class of stable isotope labels , the Basic Mass Tags (BMT). The qPST™ combines the features of relative protein quantification via isotopomeric peptides pairs and MS/MS based identification. As required for accurate performance, the corresponding qPST™-taglabelled ‘light’ and ‘heavy’ peptides show identical chromatographic retention times. A key feature of the qPST™ approach is that the labeling can be performed at the protein or large peptide level and hence sample pairs can be combined at an early stage of the analysis process. Resulting advantages to other approaches are shown. As a proof of concept study we have differentially analyzed the lysate of yeast grown on either ethanol or galactose as the only carbon source. The differential protein expression results obtained from this yeast qPST™ experiment, which will be shown in this presentation, are in agreement with the data available in the public domain. Tandem Mass Tags® (TMT®), a further tagging approach developed by our team introduces a novel and innovative concept for highly accurate and sensitive quantification of peptides/proteins in complex proteomes [4]. This is achieved by the special design of the TMT tag which consists of (i) reporter group, (ii) a mass balancing group and (iii) a reactive group. Consequently all TMT® labelled peptide pairs have the identical mass and the quantification is achieved by specific reporter ions at predicted m/z values generated in the MS/MS mode. The advantages of this approach are shown. Literature [1] Steiner, S.; Anderson, N. L. Pharmaceutical proteomics, Ann N Y Acad Sci. 2000, 919, 48-51 [2] Steiner, S.; Witzmann, F. Electrophoresis. 2000, 21, 2099-104 [3] Kuhn, K.; Thompson, A.; Prinz, T.; Müller, J.; Baumann, C.; Schmidt, G.; Neumann, T.; Hamon, C. (2003) J. Proteome Res., 2003, 2, 598-609 [4] Thompson, A.; Schäfer J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.; Neumann, T.; Hamon, C. Anal. Chem. 2003, 75, 1895-1904

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Lecture V21 Laserdesorption von Biomolekül-Ionen aus Mikrotröpfchen Nina Morgner, Hans-Dieter Barth, Bernhard Brutschy J.W.Goethe-University , Institute for Physical and Theoretical Chemistry Marie-Curiestr. 11, 60439 Frankfurt Die Massenspektrometrie (MS) wurde vor einigen Jahren durch die Methoden ESI-MS (Electro Spray Ionisation) und MALDI-MS (Matrix-Assisted Laser Desorption/Ionisation) revolutioniert. Beide Methoden haben spezifische Vor- und Nachteile und finden zunehmend Verwendung in Biologie, Biochemie und Pharmazie. Seit einigen Jahren wird in unserer Abteilung eine alternative Methode entwickelt, genannt LILBID-MS (Laser Induced Liquid Beam/Bead Ion Desorption)[1], die versucht Vorteile von beiden Methoden in sich zu vereinen. Dabei werden Biomolekül-Ionen aus Mikrotröpfchen (natürliche Matrix) mit einem gepulsten IR Laser durch resonante Schwingungsanregung der Lösungsmittelmoleküle ins Vakuum desorbiert/ablatiert. LILBID ist eine hochsensible Methode, die die Untersuchung von Biomolekülen in nativer Lösung bei niedrigem Analytverbrauch erlaubt. Sie ist tolerant gegen hohe Salzkonzentrationen und schonend genug, um die Detektion von spezifischen nicht kovalent gebundenen Biokomplexen zu ermöglichen. Bis jetzt konnten wir Makromoleküle (Antibiotika), Proteine (Lysozym), und Oligonukleotide aus wässriger Lösung nachweisen. Wir konnten nicht-kovalent gebundene Biokomplexe wie Hämoglobin oder DNA-Duplexe sowie deren spezifische Bindung mit Minor-groove-Bindern messen. Als Beispiel für spezifische RNA-Liganden-Bindung wurde der Tat-TAR-HIV regulatory Komplex gemessen. Ein großes Membranprotein, das wir detektieren konnten, ist Cytochrome C Oxidase (ca. 120 kDa). In vielen Fällen ist die Analyse eines Tröpfchens ausreichend für ein Massenspektrum, was der Methode das Potential zu einer absoluten Sensitivität im Attomolbereich verspricht.

Literatur [1] W. Kleinekofort, J. Avdiev, B. Brutschy A new method of laser desorption mass spectrometry for the study of biological macromolecules Int. J. Mass. Spectr. Ion Proc. 152 (1996) 135-142

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Lecture V22 Parallel Post Source Decay MALDI-TOF – Principles and Application to Proteomics Marten Snel1, Marc Kipping2, Jeff Brown1, Daniel Kenny1, Bob Bateman1 1

Waters Corporation MS technologies centre, Atlas Park, Simonsway, Manchester M22 5PP, UK. 2 Waters GmbH, Hauptstr. 87, 65760 Eschborn, GE.

Reflectron based MALDI-TOF mass spectrometers are able to separate and analyse fragment ions produced from precursor ions that spontaneously decompose in flight. Such ions are generally referred to as metastable ions, and the process of decomposition in the region between the ion source and the reflectron is commonly referred to as post source decay (PSD). The analysis of such PSD ions is an established technique, which is capable of providing complementary structural information. In practise, a timed electrostatic ion gate is utilised to isolate a single precursor mass and it’s associated fragments thus ensuring that all detected fragment ions are attributed to the correct precursor. This inherent requirement consequently leads to serial data acquisition and thus a long acquisition time and increased sample consumption. Additionally, low mass, and hence low kinetic energy fragments, do not fully penetrate the reflectron and are not correctly focussed onto the detector. This problem is alleviated by successively reducing the reflectron voltage in steps and acquiring additional data for each step. The focussed regions of the resultant spectra may then be “stitched” together to form a complete spectrum. We present a novel enhancement to PSD analysis by simultaneously acquiring PSD fragments from all parent ions. Fragment ions are correctly matched to their corresponding precursor ion by acquiring an additional spectrum for each conventional reflectron step at a slightly lower reflectron voltage. By measuring the difference in TOF for fragment ions produced by the change in reflectron voltage, it is possible to calculate the mass of the associated precursor ion. This new “parallel PSD” technique reduces analysis time and consumes less sample than conventional PSD. To demonstrate the application of parallel PSD in protein identification, we mass analysed 96 samples from a 2D gel separated E. coli lysate using both traditional peptide mass fingerprinting (PMF) and, from the same sample spot, parallel PSD. PSD data were acquired at 12 different reflectron steps. The 15 most intense peaks from the MS spectrum were considered as candidate parents and a data processing algorithm calculated a list of fragment masses associated for each parent. The results were searched against a SWISSPROT protein database. Using PMF we unambiguously identified 53 proteins. The number of proteins we identified using parallel PSD was 81. In total we found 83 proteins, 2 only by PMF, 30 only by parallel PSD and 51 by both techniques.

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Lecture V23 Cryodetektion mit MALDI-TOF-MS. Eine neue Methode für die Routineanalyse von großen Biomolekülen R.Woisch1 und U. Matter2 1

GSG Meß- und Analysengeräte GmbH, Im Technologiedorf 9, 76646 Bruchsal; 2Comet Analytics AG, Herrengasse 10, CH-3175 Flamatt, Schweiz Konventionelle Mikrochannelplate (MCP) Detektoren funktionieren nach dem Prinzip der Sekundärelektronenvervielfachung. Elektronen die beim Aufprall von Ionen mit hoher Geschwindigkeit freigesetzt werden setzen eine Elektronenkaskade in Gang. Aufgrund der niedrigeren Geschwindigkeit großer Moleküle ist dieser Prozess weniger effizient, so dass die MCP- Sensitivität mit zunehmender Masse abnimmt. Dieser Effekt zeigt sich bei der Analyse von intakten Proteinen, Nukleinsäuren und Polymeren. Supraleitende (Superconducting tunneling junction, STJ) Detektoren haben diese Eigenschaft nicht weil die kinetische Energie der Teilchen direkt gemessen wird und die Detektorsensitivität für alle Massen 100 % beträgt. Nachfolgend wird das erste kommerzielle STJ Massenspektrometer für den routinemäßigen Einsatz im Labor beschrieben. Der Macromizer ist ein MALDI-TOF Massenspektrometer mit einem 16-Kanal STJ Detektor und einem geschlossenem 4He-3He-Cyrostatsystem. Mit diesem Cyrostatsystem wird der Detektor auf 0,3 K gehalten. Eine bewegliche Ionenkanone wird dazu benutzt den Ionenstrahl über seine x und y Achse zum 1 mm2 großen Detektor zu lenken und einzustellen. Proteine mit sehr großer Masse können ohne Schwierigkeiten detektiert werden. Human Immunoglobulin A und M, Molekulargewicht ungefähr 400 kDa, bzw. 1000 kDa werden als einfach protonierte Ionen bei 1 pmol gemessen. Human Immunoglobulin G, Molekulargewicht zirka 150 kDa, wird als einfach protoniertes Monomer, Dimer, Trimer und Tetramer detektiert. Das letztere Ion hat eine Masse von ungefähr 600 kDa. Messungen von Multimeren des von Willebrant Faktors zeigen Peaks bis 2.1 MDa. Da die Detektorsensitivität massenunabhängig ist, repräsentiert die relative Intensität eines Peaks die wahre Menge des Ions im Ionenstrahl. Alle Detektorsignale resultieren von tatsächlichen Partikelaufschlägen. Daher sind die Spektren vom Macromizer frei von Rauschen und zeigen eine hohe Empfindlichkeit. BSA mit einem Molekulargewicht von 66,4 kDa kann mit einer Konzentration von 1 fmol als einfach geladenes Ion nachgewiesen werden. Der Macromizer generiert ein Energie vs Massen Scatterplot sowie ein Intensität vs Massen Spektrum. Ein doppelt geladenes Ion produziert die doppelte Energie eines einfach geladenen Ions gleicher Masse. Ein Energiefilter gestattet die Entfaltung von Massenspektren durch Subtraktion von Mehrfach-Ladungszuständen. Der Macromizer kann darüber hinaus auch für die Analyse von komplexen Proteinmischungen wie z.B. Serum oder Zell-Lysate eingesetzt werden. Mit Hilfe einer simplen Vorfraktionierung konnte ein weiter Bereich von Proteinen mit Massen von 3-150 kDa gemessen werden. Als Biomarker Discovery Plattform konnten wir zeigen, dass der Macromizer alternative Technologien bezüglich Massenbereich und Sensitivität bei hohen Massen übertrifft.

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Lecture V24 Validierung von Proteinidentifizierungen durch Multiple Reaction Monitoring (MRM)-getriggerte LC/MS/MS Acquisition Henning Urlaub 1, Christof Lenz 2, Reinhard Lührmann 1 1

Max-Planck-Institut für Biophysikalische Chemie, Am Faßberg 11, 37077 Göttingen 2 Applied Biosystems, Frankfurter Strasse 129 B, 64293 Darmstadt.

Der Erfolg der Massenspektrometrie auf den Gebieten der Proteinforschung und Proteomics beruht im wesentlichen auf ihrer Fähigkeit zur sensitiven Proteinidentifizierung, selbst aus komplexen Gemischen. Die Aussagekraft solcher Identifizierungen, die zumeist auf der Zuordnung einiger weniger Verdaupeptide beruht, ist dabei nach wie vor umstritten.1,2 Der Zuordnung weiterer vermuteter Verdaupeptide zur Erhöhung der Aussagekraft sind experimentelle Grenzen gesetzt: in komplexen Gemischen sind dies vor allem die selektive Detektion der Analyten vor dem Hintergrund im MS, sowie im Fall von LC/MS/MSExperimenten die MS/MS-Kapazität des Massenspektrometers. Die Selektivität der Detektion von in der Probe vermuteten Analyten kann deutlich erhöht werden, wenn statt eines MS-Übersichtsscans vorhergesagte Fragmentierungsreaktionen im MS/MS verwendet werden (MRM, Multiple Reaction Monitoring). In der vorliegenden Studie wurde hierfür ein hybrides Triple Quadrupol/Lineare IonenfallenMassenspektrometer (Q TRAP) verwendet.3 Zum Nachweis gering abundanter, erwarteter Proteine in LC/MS/MS-Analysen komplexer Gemische werden zuvor theoretische Trypsinverdaue durchgeführt. Für geeignete Peptidsequenzen werden spezifische Fragmentierungsreaktionen vorhergesagt, die in eine Liste von MRM-Übergängen übersetzt werden. Das Spektrometer wird hierbei im Triple Quadrupol-Modus betrieben. Falls ein oder mehrere Übergänge eine spezifizierte Intensität überschreiten, werden zur Bestätigung der Peptididentität im selben Zyklus Produktionenspektren im Lineare Ionenfallen-Modus ausgelöst. Jedes Peptid wird somit durch einen MRM-Übergang, seine Retentionszeit und ein volles Produktionenspektrum charakterisiert.4 Die beschriebene Methodik wurde u.A. am Beispiel von In-Gel-Verdauen spleißosomaler Proteine getestet. Spleißosomale Proteinkomplexe wurden gefällt, die Proteine auf einem 1D-Gel aufgetrennt und im Gel verdaut. In vorhergehenden Analysen identifizierte oder vermutete Proteine wurden theoretisch verdaut und in MRM-basierte LC/MS/MS Methoden umgesetzt. Vergleiche mit Targetanalysen unter Verwendung herkömmlicher Acquisitionsschemata zeigen eine deutlich erhöhte Zahl eindeutig zugeordneter Verdaupeptide, und somit eine deutlich höhere Aussagekraft der MRM-basierten Methodik. Dies ist z.B. für Biomarker-Analysen von Bedeutung. Literatur [1] M.A. Baldwin, Mol. & Cell. Proteomics 3, Perspective (2004). [2] B.J. Cargile et al., J. Proteome Res. 3, 1082-5 (2004). [3] J.W. Hager, Rapid Comm. Mass Spectrom. 16, 512-26 (2002). [4] J. Hopfgartner et al., J. Mass Spectrom. 39, 845-55 (2004).

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Lecture V25 Fast Prototyping of Hydrophobic Disposable Polymer Support Arrays for Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Proteins by Atmospheric Molding Alexander Muck and Aleš Svatoš Max-Planck-Institute for Chemical Ecology, Hans-Knöll-Str. 8, D-07745 Jena Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been since its introduction in the late 80´s widely used to determine the molecular masses of macromolecules, such as proteins and polymers [1,2]. The new material properties and specific surface coatings influencing the yield and purity of analyzed species have been studied in MALDI-MS since the emergence of the surface-enhanced laser desorption/ionization affinity technology (SELDI-MS) in the recent years [3]. Nevertheless, measurements of complex protein samples and analysis of membrane proteins still represent a challenging task. We describe a new protocol for prototyping hydrophobic disposable poly(alkyl methacrylate-co-methyl methacrylate) copolymer sample support arrays for MALDI-MS of proteins by atmospheric molding [4]. The sample support arrays were replicated by molding prepolymer alkyl methacrylate solutions into molds containing a micromachined silicon master, an aluminum-spacer and glass cover plates, followed by UV-initiated in-situ polymerization under atmospheric pressure. The fabrication procedure enabled a simultaneous fabrication/modification of single-use polymer arrays by an appropriate selection of functional groups of the copolymerized monomers during molding. The one-step modification is demonstrated for enhanced protein adsorption to the modified materials by introduction of hydrophobic butyl-, dodecyl-, and octadecyl groups to the polymer backbone without a need for additional surface coating or derivatization. The MALDI-MS performance of the new polymer chips was tested for spectral measurements of bovine pancreas insulin, horse heart myoglobin and bovine serum albumin. The protein adsorption to the new hydrophobic copolymer chips was studied for bovine pancreas trypsinogen, the sample desalting parameters such as time and volume were optimized for myoglobin as model proteins. A significant signal increase was achieved after efficient desalting of an insect Δ11-desaturase membrane protein fragment from a complex elution buffer [100 mM phosphate, 10 mM Tris(hydroxyethyl)aminomethane, 0.5 M NaCl, and 10 mM ethylenediamine tetraacetic acid] on the poly(butyl methacrylate-co-methyl methacrylate) copolymer chip (monomer ratio 8:2, v/v) by simple washing of the target zones. The new chips offer reduced sample manipulation and device fabrication times as well as simple operation. Literature [1] Karas, M., Hillenkamp, F., Anal.Chem. 1998, 60, 2299-2301. [2] Aebersold, R., Goodlett, D.R., Chem. Rev. 2001, 101, 269-295. [3] Tang, N., Tornatore, P., Weinberger, S.R., Mass Spectrom. Rev. 2004, 23, 34-44. [4] Muck, A., Svatoš, A., Rapid Comm. Mass Spectrom. 2004, 18, 1459-1464.

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Lecture V31 Die Kopplung von SEC und Q-TOF-MS offenbart den hochsystematischen Aufbau von Fulvinsäuren Anja These und Thorsten Reemtsma Technische Universität Berlin, Fachgebiet Wasserreinhaltung, Sekr KF 4, Str. des 17 Juni 135, 10623 Berlin. Fulvosäuren, eine Fraktion der Huminstoffe, machen mehr als 50% des gelösten organischen Kohlenstoffs in natürlichen Wässern aus und sind damit die mengenmäßig herausragende organischen Stoffklasse in der aquatischen Umwelt. Massenspektrometrische Untersuchungen dieser Substanzklasse gelangen lange Zeit nur nach Zersetzung (z.b. Pyrolyse-MS), erst durch die Elektrospray-Ionisation wurden intakte Moleküle zugänglich [1]. Durch geeignete Pufferzusätze zur Größenausschlusschromatographie (SEC) ist es möglich, diese Polycarbonsäuren in Monoanionen zu überführen und so die Komplexität der Massenspektren zu verringern [2]. Dennoch werden komplexe Spektren erhalten, in denen jede Masse von mehreren isobaren Molekülen mit konstanter Massendifferenz besetzt ist. Die Auflösung eines ESI-TOF-MS reicht gerade aus, diese Moleküle massenspektrometrisch zu unterscheiden und bietet somit die Möglichkeit, einzelne Fulvosäuremoleküle zu untersuchen [3]. In einem (kleinen) Massenbereich von m/z 190 bis 340 haben wir über 200 Summenformeln von Fulvosäuremolekülen bestimmt. Die Systematik in der Elementarzusammensetzung der Fulvosäuremoleküle lässt sich durch verschiedene Parameter, z. B. in einem van Krevelen-Diagramm (H/C vs. O/C), sichtbar machen. Hier nehmen die Fulvosäuren charakteristische Regionen ein. Das Auftreten der Isobaren lässt sich durch drei Homologenreihen erklären, von denen jeweils bestimmte Mitglieder in höchster Intensität auftreten. Ergänzende Einblicke in den systematischen Aufbau dieser Stoffgruppe liefern die ProduktIonen-Spektren aus Q-TOF-MS Untersuchungen. Hier werden ungewöhnlich fragmentarme Spektren erhalten, die von mehrfachen Decarboxylierung und Wasserabspaltung dominiert werden, deren Ausmaß sich exakt aus der Elementarzusammensetzung des jeweiligen Moleküls voraussagen lässt. Dieses Fragmentationsverhalten kennzeichnet die Fulvosäuren als eine in sich homogene Stoffgruppe und grenzt sie zugleich von allen anderen, uns bekannten biogenen Stoffklassen ab. Die Elektrospray-Massenspektrometrie mit TOF-MS oder noch höherer Auflösung bietet einen grundlegend neuen Zugang zur Klasse der Fulvosäuren und eröffnet jetzt die Möglichkeit, Bildung und Transformationen dieser wichtigen natürlichen Stoffgruppe auf molekularer Ebene zu untersuchen. Literatur [1] [2] [3]

McIntyre, C., Batts, B.D., and Jardine, D.R., J. Mass Spectrom., 1997. 32 (3): p. 328-330. Reemtsma, T. and These, A., Anal. Chem., 2003. 75 (6): p. 1500-1507. These, A., Winkler, M., Thomas, C., and Reemtsma, T., Rapid Commun. Mass Spectrom., 2004. 18 (16): p. 1777-1786.

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Lecture V32 MONITORING AND FATE OF POLAR PERSISTENT POLLUTANS IN WASTE AND SURFACE WATER IN EUROPE Marco Bernhard, Jutta Müller, Thomas P. Knepper, Europa Fachhochschule Fresenius, Limburger Strasse 2, D-65510 Idstein

The EU-funded project “Removal of Persistent Polar Pollutants Through Improved Treatment of Wastewater Effluents (P-THREE)” aims to achieve an improved understanding of the bio transformation and removal of polar persistent pollutants (P3) in municipal wastewater (WW) treatment. Conventional wastewater treatment plants (WWTP) are not designed to deal with polar pollutants, and, in many cases some compounds are not totally eliminated by conventional activated sludge treatment and they are discharged into surface water and in some cases can reach the drinking water. The selection of the approx. 40 P3-compounds to be monitored was based, beside their given polarity, additionally upon production rates, regulation and known tendency to even by-pass filtration steps during drinking water treatment. Some of these pollutants are acidic pesticides and pharmaceuticals because of high water solubility. In this study some representative compounds of these two group of compounds have been selected such as, the phenoxy acids (MCPP, MCPA, 2,4-D, 2,4-DP) and bentazone as representatives of acidic herbicides and analgesics diclofenac and ibuprofen as representatives of acidic pharmaceuticals. There are only a few publications discussing the biodegradation of the selected compounds in real WWTP, but in all of them the incompletely degradation of these compounds and the risk of reaching the drinking water was showed. In order to avoid and reduce these pollutants in wastewater effluents a membrane bioreactor (MBR) was tested. A 20 L pilot plant membrane bioreactor was fed with real raw water spiked with 10 g/L of the selected compounds. The spiking experiment was repeated every week during four weeks to make possible the adaptation of microorganisms. In order to study further the biodegradability of these compounds a biologically active fixed-bed bioreactor (FBBR) was used where spiked effluent wastewater was pumped continuously in a closed loop. All the samples were analysed by SPE-GC-MS. In both cases the elimination of selected compounds was reached except for the bentazone, and diclofenac in the FBBR. The presented data obtained from this comprehensive study indicate, that P3 compounds occur throughout Europe. For their removal more effective WW treatment technologies other than AST are required. Acknowledgements This work has been supported the EU Project P-THREE (EVK1-CT-2002-00116).

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Lecture V33 Analyse von Galaxolid und Tonalid mittels ’Triple-Quadrupole Linear Iontrap MS’ Claudia Martin1, Monika Möder2, Gudrun Krauß1, Dietmar Schlosser1, Roland Geyer1

1

UFZ Umweltforschungszentrum Leipzig-Halle in der Helmholtz-Gemeinschaft Department Grundwassermikrobiologie, Theodor-Lieser-Str. 4, D-06120 Halle/Saale 2 Department Analytik, Permoserstr. 15, D-04318 Leipzig

In Oberflächenwässer gelangte, bio-aktive Spurenkontaminanten können aufgrund ihres oftmals unbekannten Umweltverhaltens eine potentielle Gefahr für Mensch und Ökosysteme darstellen. Von großem Interesse ist deshalb die Aufklärung natürlicher Abbauprozesse, die das Umweltverhaltens dieser Stoffe mitbestimmen. Zwei Beispiele, solcher in aquatischen Ökosystemen nachweisbarer, organischer Spurenkontaminanten, sind die polycyclischen Moschusduftstoffe Galaxolid (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8hexamethylcyclopenta-γ-2-benzopyrane, HHCB) und Tonalid (7-acetyl-1,1,3,4,4,6hexamethyl-1,2,3,4-tetrahydro-naphthalene, AHTN) [1]. Diese Riechstoffe, Ersatzstoffe für die als kanzerogen eingestuften und verbotenen Nitromoschusdurftstoffe, finden weite Verbreitung in Kosmetikprodukten. Smital et.al. konnten für Galaxolid und Tonalid die Hemmung des „Multixenobiotic Resistance“-Abwehrsystems in aquatischen Organismen nachweisen [2]. Deshalb ist der Nachweis ihrer biologischen Abbaubarkeit von besonderer Bedeutung. In unseren Untersuchungen wurden Galaxolid und Tonalid von aquatischen Pilzkulturen abgebaut, die an verschiedenen Probennahmestandorten entlang der Saale im Stadtgebiet von Halle aus Wasser und Sediment isoliert wurden. Untersuchungen mittels GC-MS zeigten, dass dabei entstandene Metabolite zum einen durch Hydroxylierung dieser Substanzen entstanden. Die exakten Metabolitstrukturen werden derzeit noch untersucht. Unser Anliegen war es mittels eines Hybrid Quadrupole Linear Ionenfallen MS 1. die Sensitivität und Spezifität der Analyse von Galaxolid und Tonalid zu optimieren (Vergleich ESI, APCI), 2. Strukturvorschläge für Metabolite durch Vergleich von 3 MS Fragmentierungsmustern zu verifizieren und zu präzisieren sowie 3. mit automatisierter Datenanalyse (IDA) weitere Metabolite zu identifizieren [3]

[1] Buerge, IJ; Buser, HR; Muller, MD; Poiger, T. Environmental science & technology 2003, 37 (24), 56365644 [2] Smital,T.; Luckenbach,T.; Sauerborn,R.; Hamdoun,A. M.; Vega,R. L.; Epel,D. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis 2004, 552, 101-117 [3] Hopfgartner, G.; Varesio, E.; Tschäppät, V.; Grivet, C.; Bourgogne, E.; Leuthold, L. A. Journal of Mass Spectrometry 2004, 39, 845-855.

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Lecture V34 TD/GC/MS zur Atemluftanalyse Grundmann, A. 1, Nolte, J. 1,Baumbach, J.I. 1, Besche, S. 1, Bandur, R. 1 und Hoffmann, Th 2

1

2

ISAS Dortmund, Bunsen-Kirchhoff-Str. 11, D-44139 Dortmund. Johannes Gutenberg-Universität, Duesbergweg 10-14, 55128 Mainz.

Untersuchungen humaner Atemluft sind seit mehreren hundert Jahren als diagnostisches Hilfsmittel bekannt. Anhand des Geruchs der ausgeatmeten Luft können Rückschlüsse auf einige Erkrankungen gezogen werden [1]. Noch heute können einige Krankheiten mit Hilfe charakteristischer Aromen in der ausgeatmeten Luft diagnostiziert werden, ein Beispiel ist der Geruch faulender Äpfel für Patienten mit diabetischer Ketoazidose [2]. Da nicht für jedes Krankheitsbild typische Gerüche in der Atemluft wahrgenommen werden können, kommen zunehmend sensitive, analytische Verfahren zum Einsatz. Die Thermodesorption/Gaschromatographie/Massenspektrometrie (TD/GC/MS) bietet die Möglichkeit einer einfachen, schnellen, aussagekräftigen, nicht invasiven Analyse. Diese Methode hat den Vorteil, keine Nebenwirkungen zu haben und auch für schwache Patienten und Kinder einsetzbar zu sein. Die Analyse der Proben kann relativ schnell durchgeführt werden und ist im Vergleich zu herkömmlichen Untersuchungsmethoden wie Bronchoskopie oder Röntgen deutlich günstiger. Die Atemluft wird auf speziellen Adsorptionsröhrchen angereichert und mittels GC/MS aufgetrennt und analysiert. Besondere Bedeutung kommt dabei der Trocknung der Atemluft bei der Probenahme zu, um den Eintrag an Wasser in das Massenspektrometer zu minimieren. Danach sollen selektiv charakteristische Analyten für gesunde wie auch kranke Personen gefunden werden, von denen einige später als Biomarker für Krankheiten in Frage kommen. Bisher wurden leichtflüchtige Analyten wie kurzkettige Alkohole und Ketone, einige Schwefelverbindungen, Aromaten, etc. in allen Proben gefunden. Quantitative Auswertung wurde mit Hilfe einer speziell auf die Probenahmebedingungen abgestimmten Testgasquelle durchgeführt. Erste Ergebnisse liegen im ng/L Bereich, diese werden präsentiert und diskutiert. Außerdem wurde der Einfluß der Bedingungen bei der Probenahme (z.B. Ernährung, Hungergefühl, Rauchen) untersucht. Literatur [1] M. Phillips et al., J. Chromatogr. B 1999, 729, 75. [2] M. Phillips et al., Disease markers in exhaled breath 2002, New York, 219.

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Lecture V35 Schneller simultaner Nachweis von Stickoxiden: Kombination moderner Methoden mit Methoden aus dem Wissensschatz der MS-Grundlagenforschung Ulrich Boesl, Alexander Bornschlegl, Rares Vintan Technische Universität München, Department Chemie, Physikalische Chemie Stickoxide stehen in der Rangliste der Luftschadstoffe mit an oberster Stelle. Hauptquelle sind Verbrennungsmotoren. Um diese zu optimieren, müssen vor allem auch NO und NO2 simultan und sehr schnell - also mit 10-100 Millisekunden-Auflösung nachgewiesen werden. Erheblich erschwert wird dies durch die Zusammensetzung von Motor-Emissionen, in denen andere Abgaskomponenten mit teils um Größenordnungen höheren Konzentration, sowie eine große Vielzahl verschiedener chemischer Komponenten vorkommen. Konventionelle Methoden können dies nicht leisten. Dazu zählen CLD (chemische Lumineszenz Detektion), Infrarotspektroskopie oder auch Elektronenstoß-Massenspektrometrie. Selbst die für solche Fälle optimale Resonanz-Ionisation (REMPI) mit Lasern kombiniert mit Flugzeitmassenspektrometrie versagt hier wegen des sehr schnellen Zerfalls von NO2 im angeregten elektronischen Zustand. Die Fragmente sind NO und O, und damit ist im Massenspektrum NO2 nicht von primärem NO zu unterscheiden. Hier wenden wir einen kleinen Trick der massenspektrometrischen Grundlagenforschung an: Wir nützen die hohe freiwerdende kinetische Energie der Fragmente NO und O um primäres und sekundäres NO unterscheiden zu können. Im Flugzeit-Massenspektrum kann eine solche freiwerdende kinetische Energie nämlich zu einer ganz speziellen Massenpeakform führen. Dies wird in unserer Präsentation erklärt und eine aktuelle Anwendung gezeigt. Die vorgestellte Methode kann im Prinzip auch auf andere molekulare Systeme angewendet werden, bei denen schnelle Dissoziation einen eindeutigen Nachweis erschwert oder gar vereitelt. Ein Beispiel sind Nitroaromaten. Mit dem hier vorgestellten kleinen Beispiel möchten wir gerne die Diskussion über den Wissensstand und Wissensschatz massenspektrometrisch-apparativer Grundlagen wieder beleben und sie den Anwendern aus den z.B. biochemischen Wissenschaften nahe bringen. Vielleicht führt eine solche Diskussion ja dazu, dass Methoden aus der Trickkiste der Grundlagenforschung eine Renaissance in der Anwendung erleben.

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Lecture V41 High-Peformance Glycoscreening and Sequencing by chip-enhanced ESI-FTICR Mass Spectrometry Martin Froesch1, Laura Bindila1, Gökhan Baykut2, Mark Allen3, Niels Lion4, Joël S. Rossier5, Hubert H. Girault4, Jasna Peter-Katalinić1 and Alina Zamfir1. 1

Institute for Medical Physics and Biophysics, University of Münster, Germany. 2 Bruker Daltonik, Bremen, Germany. 3 Advion BioSciences, Norwich, UK. 4 Laboratoire d’Electrochimie Physique et Analytique, Ecole Polytechnique Fédérale de Lausanne, Switzerland. 5 DiagnoSwiss SA, Monthey, Switzerland. Modern achievements of microfabrication technology and robotics are currently intensively considered in the mass spectrometry field, tending to replace gradually classical ESI or MALDI ion sources. The chip-based ESI MS methods were primarily introduced to proteomics and genomics, while the applications in the glycomics field are rather poor represented. In this contribution the introduction of fully automated chip and thin polymer chip ESI/FT-ICR MS for high performance mapping and sequencing of carbohydrates is presented [1, 2]. The Advion NanoMate system has been coupled to FT-ICR MS by a coupling bracket specially designed by Bruker Daltonik, Bremen. The approach has been probed for screening, sequencing by SORI-CID MS2 and identification of O-glycosylated sialylated peptides from urine of patients suffering from Schindler’s disease. Schindler’s disease is a rare inherited metabolic disorder belonging to lysosomal storage diseases (LSDs), which leads to an abnormal accumulation of sialylated and asialo-glycopeptides and oligosaccharides of about 100 times higher concentration than in healthy controls. The automated chipESI/FT-ICR MS methodology is shown here to allow increased sensitivity and ionization efficiency as compared to the standard nanoESI/FT-ICR MS by glass capillaries. It was possible to detect a number of glycoforms previously undetectable and to determine their accurate mass. Secondly, a thin polymer microchip was coupled to FT-ICR MS and optimized in the negative ion mode for glycopeptide screening. The interface between the polymer microchip and FT-ICR MS has been constructed in-laboratory. The particular attribute of the polymer chip, to achieve an increase of ionization efficiency and sensitivity, is highlighted by a large number of major and minor glycopeptide structures detected and identified in highly heterogeneous mixtures obtained from urine matrices of patients suffering from Schindler’s disease. Glycoforms expressing various saccharide chain lengths ranging from tri- to dodecasaccharide bearing up to three sialic acid moieties could be detected and assigned according to their molecular ions under the accuracy of an average mass deviation below 6 ppm. The (-)thin chipESI/FT-ICR MS is a potent novel system for glycomic screening of complex mixtures and can be considered for general applicability in the glycoanalytical field. References [1] Froesch, M., Bindila, L.M., Baykut, G., Allen, M., Peter-Katalinić, J., Zamfir, A.; Rapid Commun. Mass Spectrom., 2004, 18, 3084-3092. [2] Bindila, L.M., Froesch, M., Lion, N., Vukelić, Ž., Rossier, J., Girault, H.H., PeterKatalinić, J., Zamfir, A.; Rapid Commun. Mass Spectrom., 2004, 18, 2913-2920.

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Lecture V42 Charakterisierung von komplexen Glykoproteinen mittels Kapillarelektrophorese- ESI-oTOF am Beispiel von Erythropoietin (EPO) Matthias Pelzing, Christian Neusüß und Uwe Demelbauer Bruker Daltonik GmbH, Permoserstr. 15, D-04318 Leipzig Der Glykosylierungsgrad steht im direkten Zusammenhang zur biologischen Funktion der Glycoproteine, welche eine große Varianz von möglichen Isoformen aufweisen. Ihre Vielfalt hängt dabei von individuellen aber auch von Umgebungsfaktoren ab. Zur Charakterisierung werden weitestgehend gelbasierte Methoden wie z.B. IEF verwendet. Die detaillierte Glykanstruktur wird durch massenspektrometrische Methoden, nach enzymatischer Spaltung der Peptide bzw. Abspaltung der Glykane selbst, zugängig. Dadurch verliert man jedoch alle Information aus welchen Glykanen sich eine spezielle Isoform zusammensetzt. Wir beschreiben eine Methode zur Charakterisierung von komplexen Isoformen von Glykoproteinen auf der Ebene des intakten Proteins. Die Methode basiert auf der Kopplung von einer effizienten Trennung mittels Kapillarzonenelektrophorese und hochauflösender Massenspektrometrie. Ribonuclease B, Fetuin (Rind) and Erythropoietin (EPO) wurden als Modellproteine ausgewählt. Im Falle von EPO werden, nach unserem Stand des Wissens, erstmalig Ergebnisse zur akkuraten Masse der isomeren Glykoformen des Erythropoietin präsentiert (1). Die für Proteintrennung sehr effiziente Kapillarzonenelektrophorese (CZE) wurde mittels eines robusten “sheath-liquid interface“ (2) an ein hochauflösendes Flugzeitmassenspektrometer gekoppelt. Die entwickelte Methode ermöglicht eine direkte Abtrennung von nichtglykosylierten Proteinen, Salzen und anderer neutraler als auch geladener Spezies, die eine Ionisierung in der Elektrosprayquelle des Massenspektrometers negativ beeinflussen. Somit entfallen komplizierte Probenaufbereitungsschritte vor der Messung. Darüber hinaus können die verschiedenen Glykosylierungsformen des intakten EPO’s aufgrund der verschiedenen Ladungen, verursacht durch unterschiedliche Sialisierungsgrade, als auch aufgrund von Größenunterschieden getrennt werden. Mit dieser Methode konnten über 135 Isoformen des rekombinanten humanen Erythropoietin im Refernzematerial von European Pharmacopeia bestimmt werden. Unterschiede im Glykosylierungsmuster von EPO-Proben verschiedener Hersteller werden gezeigt. Basierend auf der Information der exakten Molekülmasse wird eine Gesamtelementarzusammensetzung der einzelnen Isoformen angegeben. Im Falle des Fetuin aus Rind konnten 75 verschiedene Isoformen mit exakter Masse bestimmt werden. Wir gehen davon aus, daß die vorgestellte Methode eine signifikante Verbesserung für die Charakterisierung und Quantifizierung isomerer Glykoformen verschiedenster Glykoproteine darstellt. Literatur 1 C. Neusüß, M. Pelzing, U. Demelbauer in Electrophoresis., 2005, in press. 2 C. Neusüß, M. Pelzing,, M. Macht., Electrophoresis 2002, 23, 3149-3159.

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Lecture V43 Stabilization of Sialic Acid in MALDI-MS and Improvement of MS/MS Performance by Amidation Sadanori Sekiya1, Koichi Tanaka1 and Yoshinao Wada2 1

Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation, Japan 2 Osaka Medical Center and Research Institute for Maternal and Child Health, Japan

Mass spectrometry (MS) using matrix-assisted laser desorption/ionization (MALDI) is a powerful tool for the analysis of oligosaccharides, and tandem mass spectrometry (MS/MS) with collision-induced dissociation (CID) is indispensable for structural analysis of oligosaccharides. However, the MS analyses of oligosaccharides including sialic acid, or Nacetylneuraminic acid (NANA), often lead its preferential dissociation and make it impossible to obtain the abundant product ions to elucidate the complete structure. We attempted amidation for stabilizing NANA. Amidation is a method to modify carboxyl group (-COOH) by converting the carboxylic acid to amide (-CONH2) in the presence of amine constituents. Amidation with ammonium chloride composed of nitrogen isotope, 15N, in the amine constituent enabled to minimize the mass change to +0.013019 after amidation. This allows the significant reduction of accompanying complexity in a structural analysis and a database search. Amidation stabilized the glycosidic bond with NANA and suppressed its dominant dissociation which was derived from in-source decay, post-source decay and collision-induced dissociation. In addition, the suppressed dissociation considerably improved the yield of the B/Y type ions for structural analysis by MS/MS. In this presentation, we demonstrate that amidation is an effective derivatization to reinforce the structural analysis of sialylated oligosaccharides by MALDI-MS. (A) %Int. 100 90 80 70 60 50 40 30 20 10 0

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Figure 1. MS spectra of disialylated biantennary oligosaccharide with PA label by MALDI-QIT-TOF MS. (A) Unamidation (B) 15N-amidation M": Molecule of oligosaccharide amidated with 15NH4Cl. B5/Y5/Y5

B5/Y6/Y5 or

B6/Y6/Y5

B6/Y5/Y4

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Figure 2. MS/MS spectrum of [M"+Na] + in Figure 1B by MALDI-QIT-TOF MS.

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2400

Lecture V44 In-capillary proteolytic digest: Identification and/or Sequencing of hemoglobins directly from blood samples and structure elucidation of glycopeptides G. Pohlentz, S. Kölbl, M. Mormann, and J. Peter-Katalinić Institute for Medical Physics and Biophysics, University of Münster, Münster, Germany We recently developed a method for direct identification and/or sequencing of proteins by proteolytic digest in the electrospray capillary and simultaneous analysis of the resulting peptides by nanoESI MS and MS/MS [1]. This strategy was now applied to the analysis of hemoglobins (HBs) directly from diluted blood samples. An amount of blood dilution in 50% methanol corresponding to 5 nl blood was mixed with ammonium hydrogen carbonate containing 10% methanol. After adding the protease (trypsin or chymotrypsin) the mixture was transferred to the electrospray capillary and data acquisition was started. For human blood samples the peptide maps usually gave sequence coverages of >80% for both, α- (HBA) and β-chains (HBB), respectively. The method worked equally well when a dried blood droplet resuspended in 50% methanol was analysed. With sequence coverages of >80% it could be identified as human (or chimpanzee; identical sequences for both chains) blood within less than 1 h. Additionally closely related species can be differentiated by in capillary digest of their hemoglobins. As an example we analyzed blood samples from Asian (Elephas maximus) and African elephants (Loxodonta africana). Besides their obvious anatomical differences, their HBs differ by 3 amino acids in the α-chain and by 1 amino acid in the β-chain. This gives rise to “marker peptides” in the peptide maps, e.g. exchange of α49S (Asian) to α49G (African) yields doubly charged peptide molecular ions at m/z 910.43 (Asian) and m/z 895.44 (African), respectively. Since in-capillary digest allows simultaneous collision-induced dissociation (CID) of the proteolytic peptides, de-novo sequencing of to date unknown HB α- and β-chains can be performed. Thus, we obtained more than 90% of the sequences of the HBAs and HBBs of the patagonian cavy (Dolichotum patagona) and the persian gazelle (Gazella subgutturosa subgutturosa) by in capillary tryptic and chymotryptic digest of the respective blood samples, CID and de-novo sequencing of resulting proteolytic peptides. Another application for in-capillary digests is the direct analysis of glycopeptides. When ribonuclease B was subjected to tryptic degradation in the electrospray capillary a number of glycopeptides could be detected. They were identified by CID experiments as 60NLTK63 attached to Man4-8GlcNAc2 and 58SRNLTK63 linked to Man4-5GlcNAc2 residues. Our results show, that in-capillary proteolytic digest in combination with nanoESI MS and MS/MS is an excellent tool for a number of applications in (glyco)protein analyses. References [1] Pohlentz, G., Kölbl, S., & Peter-Katalinić, J. (2005) Proteomics, in press

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Lecture V45 Massenspektrometrische Analyse der temperaturabhängigen Variation und der speziesspezifischen Diversität des Lipopolysaccharides des Pesterregers Yersinia pestis B. Lindner 1, A.P. Anisimov 2, und YA. Knirel 1

Forschungszentrum Borstel, D-23845 Borstel. 2 State Research Center for Applied Microbiology, Obolensk, Russia,. 3 Russian Acadamie of Sciences, Moscow, Russia. Die Pathogenität des Pest-Erregers, Yersinia pestis, wird durch eine Reihe von Faktoren bestimmt u.a. durch sein Rau-form Lipopolysaccharide (R-LPS), welches Serum- und Antibiotikaresistenz vermittelt und als Endotoxin wesentlich an der Wechselwirkung mit dem Immunsystem des Wirtes bis hin zur Auslösung eines septischen Schocks beteiligt ist. Um Resistenzmechanismen und Pathogen-Wirt Interaktionen auf molekularer Ebene zu verstehen, wurden die LPS-Strukturen verschiedener Y. pestis Stämme bei verschiedenen Wachstumstemperaturen massenspektrometrisch analysiert. Die gewählten Temperaturen (25°C und 37°C) entsprechen dabei den Temperaturen, die diese Bakterien in ihren unterschiedlichen Wirten (Fliege und Säugetier) vorfinden. Die isolierten intakten LPS wurden zunächst mittels hochauflösender ESI FT-ICR MS untersucht. Die Spektren zeigten eine Vielzahl von Molekülionen, die die biologische Heterogenität der LPS wiederspiegeln. Durch CSD (capillary skimmer dissociation) und IRMPD-MS/MS wurden intensive Fragmentionen erzeugt, die durch eine Spaltung der labilen Bindung zwischen dem Lipoid A und dem Kernoligosaccharid induziert werden und so eindeutige Aussagen über die Heterogenität innerhalb des Lipoid A und des Kernoligosaccharids erlauben. Durch milde Säurehydrolyse des LPS und chromatographischer Fraktionierung wurden zusätzlich Oligosaccharide aufgereinigt und massenspektrometrisch identifiziert, die dann mittels NMR-Spektrometrie detailliert analysiert wurden. Während bei einer Wachstumstemperatur von 37°C nur ein Kernoligosaccharid synthetisiert wird, findet man bei 25°C hauptsächlich vier Strukturen, die sich durch unterschiedliche terminale Monosaccharide (Gal oder Hep bzw. Kdo oder Ko) in der verzweigten Zuckerstruktur voneinander unterscheiden. Dagegen zeigten die Stämme, die bei 25°C gewachsen waren, ausschließlich tetraacalyliertes Lipoid A, während auch penta- und hexaacyliertes Moleküle bei einer Wachstumstemperatur von 37°C gefunden wurden. Die Phosphategruppen im Lipoid A sind nichtstoiometrisch mit Ara4N substitutiert. Der Gehalt ist bei 37 °C deutlich geringer.

Diese Arbeit wurde im Rahmen des Int. Science and Technology Center Patner Project #1197p durchgeführt.

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Lecture V51 Entwicklung einer rasternden Atmosphärendruck (AP)-MALDI Quelle für das Finnigan LTQ-FT Massenspektrometer Martin Köstler, Dieter Kirsch und Bernhard Spengler Institut für Anorganische und Analytische Chemie, Justus-Liebig-Universität, Schubertstraße 60 / Gebäude 16, 35392 Gießen MALDI als wichtige Methode der Bioanalytik wird seit einigen Jahren in technologisch grundsätzlich unterschiedlichen Ionenquellen-Varianten kommerziell angeboten. Neben den klassischen on-axis Hochvakuumquellen sind Feinvakuum- und AtmosphärendruckQuellen, sowie orthogonale Quellen verfügbar. AP-MALDI Quellen haben den Vorteil, dass schnell zwischen unterschiedlichen Quellentypen (ESI/NSI/MALDI) gewechselt werden und zudem der Probenwechsel sehr einfach erfolgen kann. Kommerzielle MALDI-Quellen zeichnen sich ausnahmslos durch Laserfokus-Durchmesser zwischen 50 und 200 µm aus. Diese Geometrie ist zwar für die Untersuchung von Standard-MALDI-Präparationen vorteilhaft, eignet sich jedoch nicht für die ortsaufgelöste Analyse im mikroskopischen Bereich. Für die direkte Untersuchung biologischer Proben wurde in den letzten Jahren in unserer Arbeitsgruppe ein Verfahren auf der Basis einer Hochvakuum-Quelle entwickelt, welches die bildliche Darstellung der Konzentrationsverteilung von Biomolekülen erlaubt. Dieses sogenannte SMALDI-Verfahren [1] arbeitet mit einer lateralen Auflösung von etwa 1 µm an einem Flugzeit-Massenspektrometer. Der Grund für die Entwicklung einer neuen ortsauflösenden AP-MALDI-Quelle bestand darin, eine schnell wechselbare SMALDI-Quelle für API-Systeme wie QuadrupolIonenfallen, Quadrupol-oTOF- oder Ionenfallen-FT-ICR-Massenspektrometer zu schaffen. Die mit der entwickelten rasternden AP-MALDI-Quelle erreichbaren Laserfoki liegen bei Durchmessern von 3 bis 8 µm bei einer Laserwellenlänge von 337 nm. Erste Ergebnisse, die am "Finnigan LTQ FT" Massenspektrometer erzielt wurden, sind sehr vielversprechend hinsichtlich Empfindlichkeit und Aussagekraft. Das Projekt wurde gefördert vom Bundesministerium für Bildung und Forschung, Aktenzeichen 0312834A. Literatur [1] B. Spengler, M. Hubert; J. Am. Soc. Mass Spectrom. 13 (2002) 735-748.

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Lecture V52 Liquid-matrix-assisted infrared laser desorption/ionization orthogonaltime-of-flight mass spectrometry for the analysis of gangliosides directly from thin-layer chromatography plates Klaus Dreisewerd,1 Johannes Müthing,1 Andreas Rohlfing,1 Iris Meisen,1 Željka Vukelić,2 Jasna Peter-Katalinić,1 Franz Hillenkamp,1 and Stefan Berkenkamp3 1

Institut für Medizinische Physik and Biophysik, Westfälische-Wilhelms-Universität, 48149 Münster. 2 Department of Chemistry und Biochemistry, Medical Faculty, University of Zagreb, 10000 Zagreb, Croatia. 3 Sequenom GmbH, 22761 Hamburg. We present a novel method for direct coupling of high performance thin-layer chromatography (HPTLC) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of biomolecules. One of the key features of the method is the use of a liquid matrix (glycerol), which provides an efficient wetting of the silica gel and a simple and fast MALDI preparation protocol. A second is the use of an IR laser (Er:YAG), which ablates small volumes of analyte-loaded silica gel and provides a soft desorption/ionization of even very labile analyte molecules. The orthogonal time-offlight mass spectrometer (o-TOF-MS) employed in this study, finally, provides a high accuracy of the mass determination, independent of any irregularity of the silica gel surface. The potential of the method is demonstrated by the compositional mapping of a native GM3 (II3--Neu5Ac-LacCer) ganglioside mixture from cultured Chinese hamster ovary (CHO) cells. The analysis is characterized by a high relative sensitivity, allowing the simultaneous detection of various major and minor GM3 species directly from single analyte HPTLC bands. The lateral resolution of the direct HPTLC-MS analysis is defined by the laser focal diameter of -currently- ~200 m. This allows to determine mobility profiles and retention times of individual species with a much higher precision than by the optical chromatogram. The fluorescent dye primuline was, furthermore, successfully tested as a non-destructive, MALDI compatible staining agent.

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Lecture V53 An Interface for Coupling Surface Plasmon Resonance and Mass Spectrometry Experiments Jens Grote1, Nico Dankbar2, Erk Gedig2, and Simone König1 1 Integrated Functional Genomics, Interdisciplinary Center for Clinical Research Medical Faculty, University of Münster 2 XanTec Bioanalytics GmbH, Münster, Germany A strategy for combining surface plasmon resonance (SPR) biomolecular interaction analysis and matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) is reported. Both techniques are highly complementary but need separate optimization to improve their individual specificity and sensitivity. Sensor surfaces which are optimal for kinetic analysis are not well suited for MALDI-MS and vice versa. In addition, the transfer of analyte from SPR to MS is crucial and often accompanied by sample loss. To address both of these points, a bifunctional SPR fluid cell was constructed where optimized surfaces can be used for binding studies and MS simultaneously with regard to the special need of each technique (Fig. 1). The set-up guarantees that the SPR and the loading experiment for MS are performed at identical conditions. A removable pin carries the affinity-surfacebound analyte to the mass spectrometer so that handling is minimized avoiding analyte elution. Functionalized transfer pins can also be used independently of SPR for microaffinity capture (MAC)-MS. Anal. Chem., in press.

pipette tip opening for transfer pin Opening to SPR biosensor chip 50 mm Fig. 1: Modified IBIS fluid cell and SPR chip (left), schematic showing the measurement arrangment (right).

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Lecture V54 CHIP-LCMS: A NEW FORMAT AND INTERFACE FOR NANO-LCMS FOR PROTEOMICS Friedrich Mandel 1, Tom van de Goor 2, Hongfeng Yin 2, Reid Brennen 2, Dan Sobek 2 and Kevin Killeen 2 1

Agilent Technologies, Waldbronn, Germany,

2

Agilent Technologies, Palo Alto, USA

Nano-Electrospray LCMS is currently used as a sensitive technique for analysis of complex enzymatic digested proteins and peptide mixtures. Sample handling and manipulation of precious samples of femtomole levels of peptides requires special care and chemically inert flow pathways for ultimate sample preservation. In this presentation we will show the use of microfluidics based LC-chip-MS technology that integrates nano LCMS components. A polymer microfluidic chip is described that integrates several nano-LC components in a single device, such as the trapping column, the separation column as well as the nanoelectrospray interface. Integration of these features removes the need for conventional fittings that often are causes of liquid leaks. The chip is created by using laser ablation from biocompatible, solvent resistant, flexible polymer materials such as polyimide. After ablation the chip is laminated and further small details may be created into the structure such as the electrospray emitter tip. The chip is interfaced to a commercial nano-LC system and an ion trap mass spectrometer using a novel and unique valving system located near the inlet of the MS system. The integrated valve serves to connect ports and channels within the chip structure and eliminates the interconnecting tubes and fittings. The valve rotor was also microfabricated to match the low dispersion of the chip approach. Due to the accuracy and precision of the chip fabrication process, positioning is extremely repeatable, making spray tip positioning an easy task. Examples of the use of the LC-chip-MS system are shown demonstrating sensitivity down to low femtomol levels for peptide digests. Chip-LCMS was used for the analysis of 2D-gel spots as an alternative technique to MALDI-MS. Higher percent coverage in data base searching of proteins as well as the ability to identify low abundance proteins in the presence of an abundant protein in a single gel spot were observed. The chip system can also be used in combination with other separation techniques that help reduce complexity such as SCX separation or affinity methods. This approach has been used for fraction collection and sampling onto the chip or combining a capillary method with the chip approach. The chip format offers a unique valving mechanism without internal fittings. The valve interface allows for high pressure fluidic switching between multiple fluid streams on the microfluidic device decreasing both delay – and dead volume. This allows multiple columns to be switched without additional connections and complexity allowing parallel features as well as multidimensional separation options and simultaneous operation. The microfluidic format of LC-chip-MS is a new format and interface for integrated Proteomics applications.

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Lecture V55 NanoLC coupling to ESI Chip Joerg Niebel1, Gary Schulz 2, Colleen van Pelt 3, 1

Advion Biosciences Ltd., Hethersett Norwich, U.K. 2 Advion Biosciences Inc. Ithaca, N.Y., USA

The ESI Chip (8-µm i.d.) generates an electric field equivalent to that of a 2-µm pulled capillary, thus providing the high ionization efficiency and reduced ion suppression benefits of nanoESI without the drawbacks of small diameter capillaries that often plug during use. The electrospray nozzles are etched from a silicon wafer and coated with a dielectric layer that electrically insulates the fluid from the silicon. Sixteen hundred volts applied to the fluid and zero volts applied to the silicon generates a high electric field when the liquid exits the nozzle structure due to the close proximity (a few microns) of the silicon electrode. The electric field generated from the nozzle (28-µm o.d. by 8-µm i.d.) is equivalent to that from a 2-µm pulled capillary 3 mm from the mass spectrometer counter electrode and 1 kV applied to the fluid. The high electric field of the chip provides the benefits of nanoESI producing highly charged droplets and less suppression compared to electrospray at higher flow rates. We propose that ion response ratios are dependent on the electric field strength and independent of flow rate for nanoelectrospray ionization. The benefits of coupling nanoLC chromatography to the high electric field ESI Chip are described. Liquid chromatography mass spectrometry (LC/MS) is used for the separation, identification, and quantitation of complex biological samples. NanoLC enhances sensitivity for the limited sample volumes common in protein studies. NanoLC coupled with nanoelectrospray ionization (nanoESI) provides for enhanced sensitivity, ionization efficiency, and reduced ion suppression due to the highly-charged micron and sub-micron droplets formed. The nanoLC/ESI Chip data show reduced chromatographic band broadening compared to a pulled capillary. The NanoMate 100 with ESI Chip provides for easy spray optimization and automated coupling of nanoLC columns to ESI Chip nozzles. The NanoMate 100 with an ESI Chip can analyze a wide variety of compounds, and in this paper have demonstrated the analysis of peptides, phosphopeptides, and glycopeptides with good chromatographic performance and sensitivity.

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Lecture V61 Metabolites in Human Breath: Ion Mobility Spectrometers as Diagnostic Tools for Markers of Lung Diseases J.I. Baumbach 1, D. Zimmermann 1, V.Ruzsanyi 1, W. Vautz 1, L. Freitag 2 1

ISAS – Institut for Analytical Sciences, Bunsen-Kirchhoff-Str. 11, 44139 Dortmund 2 Lung Hospital Hemer, Theo-Funccius-Str. 1, 58675 Hemer.

Volatile Metabolites occuring in human exhaled air are correlated directly to different kinds of diseases. Some metabolites are biomarkers: acetone is related to diabetes, nitric acid to asthma and ammonia to hepatitis, others are arising from bacteria. In the present paper, an ion mobility spectrometer (IMS) coupled to a multi-capillary-column (MCC) as preseparation unit is used to identify and quantify volatile metabolites occurring in human breath down to the ng/L- and pg/L-range of analytes. The spectra obtained from patients suffering on chronic obstructive pulmonary disease (COPD), Tonsilitis and pneumonia are discussed in detail. Furthermore, IMS-chromatogramms of metabolites of Serratia marcescens, Enterobacter aerogenes and Escherichia coli are compared. In addition, the effect of drug delivery on patients showing Angina lateralis is presented exemplarly to show the potential of the method developed in the field of detection of pathways, effective dosage and dicision of effective time intervals to deliver pharmaceuticals. The aim of the studies is to introduce the investigation of metabolites in human breath as method for early recognition of selected diseases and monitoring of the effectivity of drug delivery on the bases of ion mobility spectrometry data.

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Lecture V62 Unraveling Cellular Lipidomes by Simultaneous Multiple Neutral Loss Scanning and Multiple Precursor ion Scanning Schwudke D., Ejsing C.S., Shevchenko A. Max Planck Institute of Molecular Cell Biology and Genitics, 01307 Dresden, Germany Biological membranes are complex ensembles of lipids and proteins. A key necessity for the understanding of membrane function is the deciphering of complex membrane lipid composition. In eukaryotic membranes the total number of individual lipid species may well exceed thousands. Two major strategies were established for profiling cellular lipodomes in the past: 1. Direct analyses of lipid extracts by Precursor Ion Scan (PIS) and Neutral Loss Scan (NLS) 2. Separation of lipid classes by Liquid Chromatography coupled to mass spectrometry. Here we outline the use of data dependent acquisition on a QSTAR Pulsar i mass spectrometer (MDS Sciex) together with tailor made software to perform simultaneous multiple neutral loss scanning and multiple precursor ion scanning. This methodology is capable for direct and specific quantification of phosphatidylethanolamines, monomethylphosphatidylethanolamines, dimethylphosphatidylethanolamines, phosphatidylserines, phosphaditylcholines and sphingomyelins in crude lipid extracts. By integrating automatic sampling using the NanoMate© HD system (Advion BioSciences) we improved the analytical throughput and reproducibility of this novel methodology.

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Lecture V63 Rapid quantification of free and esterified phytosterols in serum using APPI-LC-MS/MS J. Lembcke, U. Ceglarek, G. M. Fiedler, S. Baumann, A. Leichtle, J. Thiery Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital, Leipzig, Germany Liebigstrasse 27 04103 Leipzig Phytosterols are structurally related to cholesterol. High plasma phytosterol concentrations in patients with hereditary sitosterolemia are associated with premature atherosclerosis [1]. Recent studies suggest that even among the general population elevated plasma phytosterol concentrations may also be associated with coronary heart disease (CHD) [2]. However, extensive epidemiological studies for the assessment of phytosterols as a potential coronary risk factor were difficult to perform because of the lack of a robust high throughput analytical platform. The aim of our investigation was to develop and evaluate a rapid method for the simultaneous quantification of free and esterified phytosterols in human serum. A novel analytic platform based on liquid chromatography and tandem mass spectrometry (LC-MS/MS) was developed. Electro spray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were compared concerning their ionization efficiency on phytosterols. The effects of different solvents and additives onto the ionization were also investigated. Highest signal intensities were obtained using APPI and neat alcohol. In a simple pre-treatment step 10 µL human serum were diluted with methanol-acetonitrile mixture. After centrifugation, free and esterified betasitosterol, campesterol, brassicasterol, and stigmasterol could be simultaneously determined by the LC-MS/MS in about 7 min.The detection limits of the different phytosterols ranged between 0.24 and 0.68 µg/L. Compared with the commonly used gas chromatography-mass spectrometric method (GC-MS) the analytical sensitivity was 100 to 250 fold higher. The linear ranges were between 1 and 1000 µg/L. The within and between run imprecision ranged between 2.4 and 9.9 %. The time of sample pretreatment and analysis could be reduced from about two hours (GC-MS) to 20 minutes [3]. The new method allows a rapid and simultaneous determination of free and esterified phytosterols in serum samples. Currently, a large-scale epidemiological study in healthy volunteers and patients with CHD is under way to clarify the role of phytosterols as a coronary risk factor. Literatur [1] Salen, G., I. Horak, M. Rothkopf, J. L. Cohen, J. Speck, G. S. Tint, V. Shore, B. Dayal, T. Chen, and S. Shefer. 1985. Lethal atherosclerosis associated with abnormal plasma and tissue sterol composition in sitosterolemia with xanthomatosis. J. Lipid Res. 26:1126-1133 [2] Sudhop, T., B. M. Gottwald, and K. Von Bergmann. 2002. Serum plant sterols as a potential risk factor for coronary heart disease. Metabolism 51:1519-1521. [3] Rapid quantification of free and esterified phytosterols in human serum using APPI-LC-MS/MS. J Lipid Res. 2005 Jan;46(1):21-6. Epub 2004 Oct 16

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Lecture V64 Time-of-Flight mass spectrometry as fast detector for two-dimensional comprehensive gas chromatography (GCxGC-TOFMS): Characterization of highly complex samples in Metabolomics and environmental analysis R. Zimmermann1, W.Welthagen1, T.Gröger1, J.Schnelle-Kreis1, O.Fiehn2, R.Shellie2 1 GSF-Forschungszentrum, Inst. für Ökologische Chemie, Neuherberg, & Universität Augsburg, Analytische Chemie, Augsburg,& BIfA GmbH, Augsburg; Germany 2 University of California Davis, Davis, CA, USA

Two-dimensional comprehensive gas chromatography (GCxGC) is a powerful technique for gas chromatographic separation of several thousands of individual compounds from highly complex mixtures within an hour [1, 2]. The coupling of GCxGC to fast time of flight mass spectrometry (GCxGC-TOFMS) now enables the recording of mass spectra of these up to several thousand chromatographically resolved compounds. However, a serious problem is the analysis and interpretation of the enormous amount of mass spectral information generated by the GCxGC-TOFMS technique. In this context, we have developed new data analysis concepts which either use characteristic MS-fragmentation pattern information and two-dimensional retention time data for “substance class” assignation of observed peaks [2] or are based on pattern recognition to identify differences between samples [3]. Two application examples are discussed: Firstly the GCxGC-TOFMS method was applied for characterisation of organics in highly complex ambient particulate matter (urban aerosol) samples. In this case a substance class assignation was performed according to the MS and GCxGC properties of the analytes. Secondly we applied GCxGC-TOFMS in a metabolomic proof of concept study for the first time for metabolite characterization in mammalian tissues. By comparison of GCxGCTOFMS data of different isogenic mice strains (NZO obese and BL/6 control strain) it was possible to identify potential biomarkers which differentiate the obese NZO strain mice form the control strain. [1] P.Marriot, R.Shellie, Trends Anal. Chem. 21 (2002) 573 [2] W.Welthagen, J.Schnelle-Kreis, R.Zimmermann, J. Chromatography A 1019 (2003) 233 [3] W.Welthagen, R.Shellie, J.Spranger, M.Ristow, R.Zimmermann, O.Fiehn, Metabolomics in press

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Lecture V65 Structure assignment of isomeric 4-nonylphenols by GC-MSn M. Möder 1, C. Martin 2, R. Vinken3 and P. Corvini3 1

UFZ Centre for Environmental Research Leipzig-Halle, Department of Analytical Chemistry, Permoserstraße 15, 04318 Leipzig, Germany 2 UFZ Department of Environmental Microbiology 3 University of Technology Aachen - RWTH Aachen, Institute of Environmental Research (Biology V), Worringerweg 1, 52074 Aachen, Germany The endocrine disrupting effects of industrial chemicals such as 4-octyl- and 4-nonylphenols (t-NP) are well known. These xenoestrogens are formed and released by microbial degradation of polyphenolalkoxylates applied as industrial detergents. These technical products, as well as those of the derived alkylphenols, are extremely complex and contain a great variety of branched isomeric phenols. The structures of individual isomers and their related estrogenic receptor response are still unknown. GC - MS protocols currently used enable the separation of about twenty mostly co-eluting 4-nonylphenol isomers. GC-MS-MS (product ion scan) investigations were performed to elucidate as much as possible isomeric structures. Basis of identification was the interpretation of product ion scan spectra in respect to the branching positions within the alkyl chain of the phenols. The related structure proposals for individual isomers were supported by physico-chemical parameters and mass spectrometric fragmentation data of some reference isomers. The combination of all data enabled a classification of the 4nonylphenol isomers by groups with common structural features mainly characterized by predominant product ions and common fragmentation pathways. A more detailed structure assignment of about 30 t-NP isomers was obtained what is the basis for further effect related studies with 4-nonylphenol fractions Literatur [1] P.F.X. Corvini, M. Elend, J. Hollender, R.Ji, A. Preiss, R. Vinken, A. Schäfer, Metabolism of 4(2´,6´dimethyl-2´-heptyl)-phenol by Spingomonas sp. Strain TTNP3, Environm. Chem. Lett., published online 25 January 2005 [2] T. F. Wheeler, J. R. Heim, M. R. LaTorre, B. Janes (1997) Mass spectral characterization of p-nonylphenol isomers using high-resolution capillary GC–MS. J Chromatogr Sci 35:19–30 The work was partially supported by DFG (DFG-Graduiertenkolleg "Adaptive physiologischbiochemische Reaktionen auf ökologisch relevante Wirkstoffe").

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Lecture V71 Nutriproteomics: Health Promotion and Disease Prevention M. Affolter, G.E. Bergonzelli, L.B. Fay, E. Guillaume, J. Hau, L.V. Lopes, L. Marvin-Guy, D. Mutch, A. Panchaud, F. Raymond and M. Kussmann Nestlé Research Centre, Nestec Ltd., Lausanne, Switzerland. Nutrition has evolved from providing nutrients to nourish populations to improving health of individuals through diet. Modern nutritional research focuses on health promotion and disease prevention, on protection against toxicity and stress, and on performance improvement. The concept of developing nutritionally enhanced or functional food requires (a) the understanding of the mechanisms of prevention and protection, (b) the identification of the biologically active molecules and (c) the demonstrated efficacy of these molecules. The major challenges for –omics in nutrition and health still lie ahead of us, some of which apply to –omic disciplines in general while others are specific for –omic discovery in the food context: (i) the integration of gene- and protein expression profiles on the one and metabolic fingerprints on the other hand is still in its infancy; we need to understand how to (a) select relevant sub-sets of information to be merged, and (b) resolve the issue of the different time-scales, at which transcripts, proteins and metabolites appear and act; (ii) the definition of health and comfort is less of a clear-cut case than the one of disease; (iii) omics in nutrition must be particularly sensitive: it has to reveal rather many weak than a few abundant signals to detect early deviations from normality; (iv) in the food context, health cannot be uncoupled from pleasure, that is, food preference and nutritional status are interconnected. Proteomics in the context of nutrition and health has the potential to (a) deliver biomarkers for health and comfort, (b) reveal early indicators for disease disposition, (c) assist in differentiating responders from non-responders in dietary terms, and, last but not least, (d) discover bioactive, beneficial food components. Independent of the context of application, proteomics represents the only platform that delivers not only markers for disposition or condition but also targets of intervention: the only way to intervene in a biological condition and to modulate its outcome is interfering with the proteins involved. In order to address some of these challenging objectives, the Nestlé Research Centre has embarked on (a) protein discovery in human milk [1,2], (b) membrane proteomics of beneficial gut bacteria (probiotics) [3], (c) identification of nutrient transporter proteins in the gut [4] and (d) finding biomarkers for intestinal stress [5]. Literature [1] Panchaud, Affolter, Kussmann; 2003 SPS Congress Applied Proteomics, Basel, Switzerland: “Analysis of protein complexes in human milk by multidimensional nanoLC-MS/MS”. [2] Panchaud, Kussmann, Affolter; Proteomics 2005, in press: “Rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by MudPIT technology”. [3] Affolter, Parche, Kussmann; 2003 SPS Congress Applied Proteomics, Basel, Switzerland: “Analysis of bacterial membrane proteins by combining low resolving SDS-PAGE with high resolving 2D-LC-MS/MS”. [4] Mutch, Anderle, Fiaux, Mansourian, Vidal, Wahli, Williamson, Roberts; Physiol. Genomics 2004 (17) 1120: “Regional variation in ABC transporter expression along the mouse intestinal tract”. [5] Marvin-Guy, Lopes, Affolter, Courtet-Compondu, Wagnière, Bergonzelli, Fay, Kussmann; Proteomics 2005, in press: “Proteomics of the rat gut: analysis of the myenteric plexus-longitudinal muscle preparation”.

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Lecture V72 Proteome Analysis of Body Fluids from Children Suffering from Juvenile Idiopathic Arthritis and Comparison to Rheumatoid Arthritis from Adults Shows Differences in Calprotectin Expression K. Falk1, S. Drynda2, C. Koy1, I. Prüfer1, S. Mikkat1, R. Nowack3, J. Oppermann3, H.-J. Thiesen4, J. Kekow2, and M.O. Glocker1 1

Proteome Center Rostock, Medical Faculty, University of Rostock, Rostock, Germany Clinic of Rheumatology, University of Magdeburg, Vogelsang-Gommern, Germany 3 Carl-Thiem-Klinikum Cottbus, Cottbus, Germany 4 Institute of Immunology, Medical Faculty, University of Rostock, Rostock, Germany 2

Juvenile Idiopathic Arthritis (JIA) is the most prevalent chronic inflammatory disease of childhood [1]. Its occurrence is very heterogeneous, although there are six distinguished subgroups. Two of them, the Seropositive Polyarthritis and the Enthesitis-related Arthritis display the most similarities with Rheumatoid Arthritis (RA) and Spondylarthropathy of adults. All mentioned diseases have in common, that due to persisting inflammation severe joint damages in late disease stages occurr, that particularly in children result in life-long remaining serious health problems. The JIA patients in our study were treated with standard therapy and assigned predominantly to the oligoarthritis subgroup. We investigated proteomes of plasma and synovial fluid [2] from eight patients suffering from JIA in a differential manner and further compared these results with samples from five adult RA patients. Pattern analysis with digitalized gel-images showed distinct and reproducible differences in spot intensities. MALDI-TOF peptide mass fingerprinting identified the following differentially expressed proteins: Vitronectin precursor and complement factor C4 were found with more abundance in plasma. Fibrinogen-β degradation products, stromelysin-1 (MMP3), ASPIC, calprotectin (MRP8/14) and protein-S precursor were identified as more abundant in synovial fluid. Fibrinogen-γ degradation products were found shifted to the acidic side in synovial fluid. The mentioned qualitative differences were detected in all JIA and RA patients with only little variations. Most interestingly, the quantitative comparison of calprotectin levels between JIA and RA patients by ELISA, showed clear differences. Calprotectin levels of adults (plasma: 14.43±2.7 µg/ml; synovial fluid: 475±58.4 µg/ml, mean+SEM) [3] are much more elevated than those of children (plasma: 7.18±2.98 µg/ml; synovial fluid: 110.40±64.46 µg/ml). The correlation of calprotectin levels and severity of inflammation let us suppose that JIA patients show only little inflammation-related tissue degradation. We found that the calprotectin levels in plasma samples from children are comparable to those from an RA subgroup which is addressed as responders to treatment with TNF-α-blockers (8.03±1.83 µg/ml) [3]. Hence, it is tempting to speculate that an early immune-modulating anti TNF-αtherapy of JIA patients should be clinically highly successful. Literature [1] K. Minden, M. Niewerth, J. Listing, A. Zink. Z. Rheumatol. 2002, 61, 616. [2] A. Sinz, M. Bantscheff, S. Mikkat, B. Ringel, S. Drynda, J. Kekow, H.-J. Thiesen, and M. O. Glocker. Electrophoresis 2002, 23, 3445. [3] S. Drynda, B. Ringel, M. Kekow, C. Kühne, A. Drynda, M.O. Glocker, H.-J. Thiesen and J. Kekow. Path. Res. Pract. 2004, 200, 165.

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Lecture V73 A standardized approach of proteome profiling of human serum using magnetic bead separation and matrix assisted laser desorption/ionizationtime of flight mass spectrometry Sven Baumann, Uta Ceglarek, Georg Martin Fiedler, Jan Lembcke, Alexander Leichtle, Joachim Thiery Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital, Leipzig, Germany

Background: The goal of using mass spectrometry (MS) in clinical proteomics is to generate proteome profiles that reflect changes in a disease state. The approach of magnetic bead purification for the analysis of low abundance proteins in human blood and other body fluids facilitates the identification of potential new diagnostic biomarkers for assessing health and disease with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The aim of our study was to establish a precise proteome fractionation technique and to validate a standardized blood sampling, processing and storage procedure for proteomic pattern analysis. Methods: The recently introduced magnetic bead separation technique was applied for the proteome profiling of human blood using MALDI-TOF-MS (mass range 1000-10000 Da). The effect of different pre-analytical conditions on the quality and reproducibility of the proteome analysis (e.g. anticoagulants, clotting, storage, temperature, and freeze-thaw cycles of the samples) was studied. Results: The proteome pattern of human serum was characterized by approximately 350 signals in the mass range of 1000-10000 Da. The proteome profile showed time dependent dynamic changes before and after centrifugation of the blood specimens. Differences of serum mass patterns were observed in the comparison of native and one-time frozen samples. The best reproducible proteomic patterns were generated after one time thawing of frozen serum samples. Conclusion: The application of the standardized pre-analytical blood sampling and storage procedure in combination with magnetic bead based fractionation allows a rapid and precise analysis of proteome patterns in human serum using MALDI-TOF-MS. This first evaluation of the pre-analytical conditions of proteome profiling in serum samples provides an opportunity to use this analytical platform for the identification of potential blood biomarkers running large scale clinical studies in patients with e.g. cancer diseases, coronary heart disease and also for experimental animal models of atherosclerosis.

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Lecture V74 Scoring proteomes by proteotypic peptides and mass spectrometry Markus Schirle 1, Parag Mallick 2, Ruedi Aebersold 2,3, and Bernhard Kuster 1 1

Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany. 2 Institute for Systems Biology, 1441 N. 34th St., Seattle, WA 98103, USA, 3 Institute for Molecular Systems Biology, ETH Hönggerberg HPT E 78, Wolfgang Pauli-Str. 16, CH-8093 Zürich, Switzerland. As it made sense in geography to first chart out the globe and then to learn how to navigate it using the resulting maps, it also seems sensible to first chart out genomic space and then learn how to navigate it. Indeed, genomic technologies have undergone a transition from a discovery phase, in which the genomic universe was charted out, to a scoring phase, in which groups of genes are identified that are correlated with specific biological conditions. Such a transition is also necessary, feasible and imminent for proteomics to achieve its potential as a genomics technology focused at the functional level. In common MS-based proteomic methods, proteins are first digested with trypsin, and the resulting peptides are analysed by LC–MS/MS. However, the peptide mixtures generated are typically extraordinarily complex. This overwhelms even the most modern mass spectrometers and results in an under-sampling of complex peptide mixtures, limits the complexity of a proteome that can be analysed; inflates the time required to carry out such analyses; and complicates the informatics challenge. It is doubtful that incremental improvements in the technology will enable high-throughput proteome analysis. We argue that the problem could be overcome by moving proteomics from a discovery to a scoring mode of operation. The datum for proteome scoring should be a peptide, more specifically, a proteotypic peptide — that is, an experimentally observable peptide that uniquely identifies a specific protein. We further suggest that the platform for proteome scoring should be an ordered array of proteotypic peptides that can be analysed by a (tandem) mass spectrometer. We have identified proteotypic peptides for many thousands of proteins by collecting peptideidentification data empirically from thousands of LC-MS/MS and MALDI MS experiments and to search that data for commonly observed peptides that uniquely identify a protein. From a set of ~500 biophysical properties of amino acids, we identified those that best distinguish observed and not observed peptides. This enabled us to develop an algorithm that can predict whether or not a peptide would be observed for any protein. Hence, sets of synthetic isotope-labeled proteotypic peptides representing proteins of interest (say human serum) can be added to proteomic experiments and function as internal reference standards for the unambiguous detection of a particular set of proteins (using the isotope signature) and the quantitative determination of the abundance of the scored proteins. In this proteome scoring method, the mass spectrometer is focused on the targeted analysis of the information-rich proteotypic peptides. Therefore, the redundancy in data collection inherent in discovery projects is eliminated, the instrument is more productively used, and the analysis of the collected data is significantly simplified. Current MALDI instruments can be equipped with kHz lasers, which allow a proteome that is arranged in an array of peptides to be scored in just a few minutes. References [1] Kuster, Schirle, Mallick & Aebersold (2005) Nat. Rev. Cell Mol. Biol., in press.

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Lecture V75 Novel MS based Strategies for Differential Quantitative Analysis of Complex Proteomes Jürgen Schäfer, Christian Baumann, Karsten Kuhn, Stefan Kienle, Christian Hamon, Sandra Steiner, Josef Schwarz Proteome Sciences R&D GmbH&CoKG, Altenhöferallee 3, 60438 Frankfurt am Main The growing demand for novel and tailored therapeutic targets and reliable biomarkers for the diagnosis, prognosis and monitoring of diseases continues to stimulate innovations in "omics" technologies. In the past decade, new insights into biochemical pathways disturbed by pathologies could be gained by differential profiling of proteins using approaches such as 2D-gelelectrophoresis [1] combined with mass spectrometry. A number of candidate targets and biomarkers have already been delivered to the drug discovery pipelines [2]. Intensive efforts have been made to develop alternative technologies for quantitative differential gelindependent proteome analysis. These techniques include promising concepts involving stable-isotope labeling combined with mass spectrometry. We have developed a gelindependent peptide/protein tags for differential quantitative proteome analysis including the quantitative Protein Sequence Tag® (qPST™) and the Tandem Mass Tag® (TMT®) technology. The qPST™ is a further development of our previously published PST® procedure [3]. The method is based on N-terminal labelling of cyanogen bromide cleaved proteins using an unique class of stable isotope labels , the Basic Mass Tags (BMT). The qPST™ combines the features of relative protein quantification via isotopomeric peptides pairs and MS/MS based identification. As required for accurate performance, the corresponding qPST™-taglabelled ‘light’ and ‘heavy’ peptides show identical chromatographic retention times. A key feature of the qPST™ approach is that the labeling can be performed at the protein or large peptide level and hence sample pairs can be combined at an early stage of the analysis process. Resulting advantages to other approaches are shown. As a proof of concept study we have differentially analyzed the lysate of yeast grown on either ethanol or galactose as the only carbon source. The differential protein expression results obtained from this yeast qPST™ experiment, which will be shown in this presentation, are in agreement with the data available in the public domain. Tandem Mass Tags® (TMT®), a further tagging approach developed by our team introduces a novel and innovative concept for highly accurate and sensitive quantification of peptides/proteins in complex proteomes [4]. This is achieved by the special design of the TMT tag which consists of (i) reporter group, (ii) a mass balancing group and (iii) a reactive group. Consequently all TMT® labelled peptide pairs have the identical mass and the quantification is achieved by specific reporter ions at predicted m/z values generated in the MS/MS mode. The advantages of this approach are shown. Literature [1] Steiner, S.; Anderson, N. L. Pharmaceutical proteomics, Ann N Y Acad Sci. 2000, 919, 48-51 [2] Steiner, S.; Witzmann, F. Electrophoresis. 2000, 21, 2099-104 [3] Kuhn, K.; Thompson, A.; Prinz, T.; Müller, J.; Baumann, C.; Schmidt, G.; Neumann, T.; Hamon, C. (2003) J. Proteome Res., 2003, 2, 598-609 [4] Thompson, A.; Schäfer J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.; Neumann, T.; Hamon, C. Anal. Chem. 2003, 75, 1895-1904

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Lecture V81 Untersuchungen der konformeren Anordnungen von Tryptophan und der Lebensdauern der zugehörigen angeregten elektronischen Zustände mit Hilfe der REMPI-TOF-MS. Zekeriya Altuğ1, Angela Gaber2 und Jürgen Grotemeyer3 1

Inst. f. Phys. Chem. der CAU zu Kiel, Olshausenstr. 40, 24098 Kiel, [email protected] 2 Inst. f. Phys. Chem. der CAU zu Kiel, Olshausenstr. 40, 24098 Kiel, [email protected] 3 Inst. f. Phys. Chem. der CAU zu Kiel, Olshausenstr. 40, 24098 Kiel, [email protected]

Aminosäuren treten in verschiedenen Konformeren auf, die eine wichtige Rolle für ihre Energie und ihre dynamischen Eigenschaften spielen. Da Aminosäuren wichtige Bestandteile von Peptidketten und Proteinen sind und starken Einfluss auf ihre Stabilität haben, ist die Untersuchung der verschiedenen Konformere sehr wichtig. Die Kenntnis ihrer Häufigkeit und der zugehörigen Energien, sowie ihrer Stabilität kann Aufschluss über ihr Verhalten in Peptidketten und somit über die Stabilität der Kette selbst geben. Mit Hilfe der Mehrphotonenanregung ist es möglich, Moleküle zu ionisieren, deren Energie eigentlich im Vakuum-UV-Bereich liegt. Die Ionisation solcher Moleküle mit Einphotonenanregung (VUV) ist nur mit großem Aufwand möglich. Mit der REMPI (Resonance Enhanced Multi Photon Ionisation)-Spektroskopie können mittels frequenzvariabler Laser im UV-Bereich elektronische und vibratorische Zustände in Molekülen bestimmt werden. Mit der resonanten 1C2P (1 Farben 2 Photon)-Ionisation können die 0-0-Übergänge der angeregten elektronischen Zustände der Konformere und ihre Energien spektroskopisch untersucht werden. Wendet man die 2C2P (2 Farben 2 Photon)-REMPI Methode an, erhält man die Ionisationsenergie der Moleküle. Mit der Pump und Probe Technik können mit der 2 Farben Anregung zudem die Lebensdauern einzelner elektronischer Übergänge gemessen werden. Hier werden die konformeren Anordnungnen von Phenylalanin und Tryptophan mit der 1C2P- und der 2C2P-Ionisation vorgestellt. Dabei wird die zu untersuchende Aminosäure aus fester Probe durch einen IR-Laser desorbiert und das Spektrum wird aufgenommen. Es werden die 6 Konformere von Tryptophan in einem Bereich von 285,5 nm bis 287 nm detektiert und die zugehörigen Lebensdauern ihrer elektronischen Übergänge vorgestellt und diskutiert. Auffällig ist, dass die Lebensdauer für zwei Konformere wesentlich höher ist, als die der anderen vier Konformere. Erste Rechnungen der Schwingungen des Zwischenzustandes zeigen, dass die Lebensdauer mit dem Schwingungsverhalten des Konformers zusammenhängt.

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Lecture V82 Peptide identification using peptide fragment ion vectors Davide Cittaro, Dario Borsotti, Alessio Maiolica, Elisabetta Argenzio, Juri Rappsilber The FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy Peptide identification using fragmentation spectra is based on considering as much information as possible. We here explore the suitability of few fragment ions to identify peptides. A new method building on attribute vectors recently described by Halligan et al. [1] was employed. We tested different combinations of small ion masses, which are readily available from fragmentation spectra. Peptides are represented as n-dimensional vectors, where n is given by the number of fragment ions considered and the peptide mass. We show that this approach, using minimal information, allows for precise and fast peptide identification. Peptide database Peptide space Calculation of coordinates and space clustering

Space querying and peptide identification

MS/MS spectrum

VLAPT R

Figure 1. A peptide sequence database is converted into a set of coordinates composed of the peptide mass and selected fragment masses. The space is then clustered using k-means algorithm. Peptides are identified extracting corresponding ions from the MS/MS spectra and finding the closest vector in the peptide space. Literatur [1] Halligan, B. D.; Dratz, E. A.; Feng, X.; Twigger, S. N.; Tonellato, P. J.; Greene, A. S. J. Prot. Res. 2004, 3, 813-820

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Lecture V83 Kompositionsanalyse und de Novo-Sequenzierung von Peptiden Bernhard Spengler, Dieter Kirsch, Vinh An Thieu Institut für Anorganische und Analytische Chemie, Justus-Liebig-Universität Gießen Schubertstraße 60, Haus 16, 35392 Gießen Mit der Verfügbarkeit hochgenauer massenspektrometrischer Daten ist die exakte Masse von Biomolekülen zu einer wichtigen physikalischen Größe in der Bioanalytik geworden. Die MALDI/ESI-FT-ICR-Massenspektrometrie, kombiniert mit einem vorgeschalteten Massenspektrometer zur Ionenmanipulation, erlaubt es auf einfache Weise, die Aminosäurezusammensetzung von mittelgroßen unbekannten Peptiden zu bestimmen, wenn die Daten aus MS- und MS/MS-Messungen kombinatorisch ausgewertet werden. Diese neue Methode, die dann in einem zweiten Schritt die zuverlässige Sequenzbestimmung der Peptide ermöglicht ("Composition-Based Sequencing (CBS)" [1]), eröffnet ein weites Feld der Anwendungsmöglichkeiten im Proteomics-Bereich. Die CBS-Methode hat eine Reihe von Vorteilen gegenüber bekannten de NovoSequenzierungsstrategien, da auf Annahmen über Zerfallsmechanismen oder Peptidspezifisches Ionisierungs- oder Fragmentieungsverhalten verzichtet werden kann. Während klassische Verfahren typischerweise die Anwesenheit erwarteter Fragmentionen-Signale überprüfen, beruht der entscheidende Schritt von CBS auf einer Auswertung der beobachteten exakten Massen hinsichtlich möglicher Zusammensetzungen der zugrundeliegenden Peptide oder Peptidfragmente. Die Möglichkeiten und Grenzen dieses Prinzips werden für typische Anwendungsfälle in Zusammenhang mit zu erwartenden Entwicklungen der Gerätetechnologie diskutiert. [1] B. Spengler, De Novo Sequencing, Peptide Composition Analysis and Compositionbased Sequencing: A new Strategy Employing Accurate Mass Determination by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J Am Soc Mass Spectrom, 15 (2004) 704-715.

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Lecture V84 Combined strategy for analysis of complex glycoconjugate mixtures from urine of CDG patients by HPAEC-PAD and mass spectrometry with computational analysis Vakhrushev, S.Y., Peter-Katalinić, J. Institute for Medical Physics and Biophysics, Biomedical Analysis, University of Muenster, Germany

Inherited metabolic diseases defined as congenital disorders of glycosylation (CDG) caused by diminished activity of enzymes, transporters or other functional proteins responsible for the glycosylation processing pathway of glycoconjugates, were described according to their clinical symptoms for the first time in 1980 [1]. For structural and diagnostic investigations of aberrant glycosylation in CDG patients with clinical symptoms plasma has been usually used to monitor the structural heterogeneity. In the urine of CDG patients, according to preliminary investigations, however, free glycans, glycosylated amino acids and glycopeptides are largely accumulated [2]. Due to this fact a high degree of heterogeneity concerning the type of the glycoconjugates, their number and the m/z values of their molecular ions can be expected in MS analysis, possibly relevant to provide structural basis for diagnostic considerations [3, 4]. Samples from urine of two CDG patients were prepared by gel permeation chromatography [2] and submitted to glycoscreening. In the fraction M3 from urine of the patient AL up to 50 different molecular ions were detected either by the negative ion mode MALDI-TOF or by capillary-based nanoESI-QTOF MS. For more detailed analysis, fraction has been submitted to high-performance anion-exchange chromatography with pulsed amperometric detector (HPAEC-PAD) separation and collected subfractions investigated by MS. As a tool for identification of already known and of still unknown components, computer algorithm for a rapid distinction of aglycon-linked glycoconjugates and free oligosaccharides has been developed. Applying the computation for assignment of molecular ions according to their m/z values allowed the monosaccharide building block and aglycon composition proposal. By this approach, it was possible to detect additional, mostly minor, components, not previously present, and to assign new structures. References: [1] Jaeken, J., Pediat. Res. 1980, 16, 179. [2] Nahrings, C., Diploma Thesis, 1994, University of Bonn, Bonn, Germany. [3] Zamfir, A., Vakhrushev, S., Sterling, A., Niebel, H.-J., Allen, M., Peter-Katalinić J., Anal. Chem. 2004, 76, 2046-2054. [4] Vakhrushev S.Y., Zamfir A., Peter-Katalinić J., J Am Soc Mass Spectrom. 2004, 15(12), 1863-8. Acknowledgements: Partial financial support of the Interdisciplinary Clinical Research Centre (IZKF) (project H1 to J. P.-K) and Sonderforschungsbereich 492 (SFB 492, project Z2 to J. P.-K.) is gratefully acknowledged.

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Lecture V91 Neue Möglichkeiten zur Erhöhung der Empfindlichkeit: Das API 5000TM LC/MS/MS Systems in der Quantifizierung kleiner Moleküle Axel Besa1, Kristin von Czapiewski1, Michal Svoboda1 1

Applied Biosystems, Darmstadt

Für den Großteil der heutigen Applikationen erscheint der derzeitige Stand der Technik im Bereich MS/MS Systemen mit den sich daraus ergebenden Nachweisgrenzen in vielen Bereichen als hinreichend. Dies liegt zum einen an den ausreichend hohen Konzentrationen der jeweiligen Analyten in den zu untersuchenden Matrices und/oder an der guten Ionisierbarkeit spezieller Analyten. Zeigt sich jedoch die Konzentrationen in den biologischen Matrices als zu gering oder wird die Ionisierbarkeit des Analyten zudem durch die Matrix negativ beeinflußt, so kann oft nur eine aufwendige, zeitintensive und damit teure Probenvorbereitung und –anreicherung zur Lösung des analytischen Problemes beitragen. Insbesondere Applikationen aus dem Bereich der pharmazeutischen Forschung, wie z.B. die Quantifizierung von Metaboliten in der Entwicklung neuer Wirkstoffe, zeigen immer wieder die Grenzen der Empfindlichkeit derzeitig etablierter LC/MS/MS Systeme. Durch Matrix hervorgerufene Effekte der Analytsuppression könnten häufig durch einen einfachen Verdünnungsschritt deutlich minimiert werden. Da dabei auch der Analyt einer Verdünnung unterliegt, verschiebt sich seine detektierbare Konzentration oftmals in den Bereich des LOD. Eine ähnliche Problematik ergibt sich nicht selten in der direkten Analytik von Rückständen, beispielsweise in Trinkwasser, wo gewünschte Zielkonzentration und LOQ oftmals zusammenfallen. Das API 5000TM LC/MS/MS System setzt neue Maßstäbe bei der Quantifizierung kleiner Moleküle in allen Bereichen der LC/MS/MS Analytik. Der Empfindlichkeitsgewinn, gemessen an der Nachweisempfindlichkeit derzeitiger LC/MS/MS Systemen, liegt analytabhängig bei einem Faktor 5 bis 15 und bildet damit die Grundlage zur Erschließung neuer Applikationsbereiche. Beispielhaft soll an verschiedenen etablierten Applikationen aus den Bereichen Lebensmittel-/Rückstandsanalytik, klinische sowie pharmazeutische Analytik die neue Grenze der Empfindlichkeit dargelegt werden: 1. Lebensmittelanalytik: Multipestizid Analytik direkt aus Trinkwasser 2. Rückstandsanalytik: Estrogene in Kläranlagenzu-/abläufen 3. Klinische Analytik: Um den Faktor 10 bis 15 gesteigerte Nachweisempfindlichkeit in der Quantifizierung von 14 Gallensäuren 4. Metabolismus: Quantitative Analytik von Nikotin und wichtigen Metaboliten

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Lecture V92

Multi Target Screening Analyse mit QTrap™ LC-MS/MS und Bibliotheksuche zum Nachweis von 300 forensisch relevanten Drogen in Körperflüssigkeiten S. Dresen1, J. Kempf1, A. Schreiber2, W. Weinmann1 1

Universitätsklinikum Freiburg; Institut für Rechtsmedizin; Albertstraße 9; 79104 Freiburg. 2 Applied Biosystems; Frankfurter Straße 129B; 64293 Darmstadt

Multi Target Screening (MTS) ist ein Information Dependent Aqusisition (IDA) Verfahren zur schnellen Detektion und Identifizierung von toxikologisch relevanten Drogen in Urin, Serum und Blut. Durch Flüssig-Flüssig- bzw. Festphasenextraktion aufbereitete Proben wurden mit einem QTrap™ LC-MS/MS–System und einer TurboIonSpray® Ionenquelle untersucht. Durch das Triple Quadrupol MS mit linearer Ionenfalle ist es möglich, sehr schnell hochempfindliche Produktionenspektren von Substanzen aufzunehmen, unmittelbar nachdem sie im MRM Modus erfasst worden sind. Die mit drei verschiedenen Kollisionsenergien aufgenommenen Produktionenspektren werden mit einer Spektrenbibliothek verglichen. Somit können 300 forensisch relevante Drogen anhand ihrer Fit-Werte durch eine Injektion identifiziert werden. Neben Realproben wurde das Verfahren auch mit aufgestockten Plasmaproben getestet, um die Zuverlässigkeit des Verfahrens und der Bibliotheksuche bei toxischen, aber auch therapeutischen Konzentrationen zu untersuchen. Dynamic Background Substraction, eine neue Funktion zur Minimierung des Hintergrundrauschens, wird vorgestellt und Vorteile für die Detektion koeluierender Substanzen im Vergleich zur Dynamic Exclusion Time aufgezeigt. MTS kann auch für die Analyse kleinerer Substanzgruppen zur Bestätigung immunologischer Vortests verwendet werden, was anhand einer neuen IDA Methode zur Bestimmung von 32 Benzodiazepinen in therapeutischen Konzentrationen gezeigt wird. Diese Art der gezielten Bestätigungsanalyse stellt eine weitere wichtige Applikation des Verfahrens in der forensischen Toxikologie dar.

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Lecture V93 Bedeutung der Flugzeitmassenspektrometrie als HPLC-Detektor bei forensisch-toxikologischen Untersuchungen Stefan W. Toennes, Moritz Wagner, Stefanie Iwersen-Bergmann, Gerold F. Kauert

Institut für Forensische Toxikologie, Zentrum der Rechtsmedizin, Kennedyallee 104, D-60596 Frankfurt/Main Fall In einem Fall vermuteter Brandstiftung wurden zwei Personen angetroffen. Die Blutprobe eines Verdächtigen, der deutliche Zeichen einer zentralnervösen Dämpfung zeigte, sollte analysiert werden, die zusätzlich entnommene Urinprobe nur asserviert werden. Untersuchungen Die Blutanalyse mittels Enzymimmunoassay gab keinen Hinweis auf Fremdstoffe, eine HPLC-DAD Screening-Analyse ebenfalls nicht. Der zuständige polizeiliche Ermittler teilte Wochen später mit, es hätte sich um das Opfer eines erweiterten Suizids nach Verabreichung von KO-Mitteln gehandelt. Die dann beauftragte Urinuntersuchung mittels GC-MS führte zum Nachweis der Aufnahme von Flunitrazepam und Levomepromazin. Die Blutprobe wurde daraufhin mittels Agilent LC/MSD TOF untersucht. Es fanden sich wirksame Konzentrationen von Levomepromazin und Flunitrazepam, die die Symptomatik und die Fallumstände erklären konnten. In einer später entnommenen Haarprobe konnten die Substanzen nicht nachgewiesen werden. Diskussion Flunitrazepam und Levomepromazin sind sehr niedrig dosierte Psychopharmaka mit sehr niedrigen wirksamen Serumkonzentrationen. Mit dem LC/MSD TOF-System (ElektrosprayInterface) gelang es, in Serum- und Haarmatrix die gesuchten Substanzen sehr empfindlich nachzuweisen. Die Extraktion der jeweiligen Feinmassen führte zu einer ausgezeichneten Selektivität für die gesuchten Substanzen selbst in Gegenwart hoher Matrixbelastung (Haare). Durch Verwendung der berechneten Feinmassen postulierter Metabolite war auch der Nachweis von z.B. mehreren oxidierten Levomepromazin-Spezies möglich, allerdings derzeit ohne strukturelle Differenzierung der verschiedenen Isomere. Schlußfolgerungen Probenmaterialien im forensisch-toxikologischen Untersuchungsgut sind üblicherweise stark matrixbelastet und erfordern eine hohe Selektivität der analytischen Methode. Die Flugzeitmassenspektrometrie als HPLC-Detektor zeigte sich in diesem Einsatzfeld als sehr leistungsfähig. Das Agilent LC/MSD TOF erfüllte die Anforderungen, die an ein einfach zu bedienendes und empfindliches Screeningsystem gestellt werden: Einzelsubstanzen können durch die Feinmasse mit hoher Selektivität nachgewiesen werden ohne dass der Bereich der in Frage kommenden Verbindungen eingeengt werden musste, wie z.B. bei MS-MSSystemen. Es bleibt noch zu zeigen, ob die Identifizierung über Feinmassenbibliotheken eine Alternative zur Identifizierung über Fragmentspektren darstellen kann.

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Lecture V94 Untersuchungen zum Alterungsverhalten von Kugelschreibereinträgen D. Kirsch1, C. Weyermann1, F. Köhler2, B. Spengler1 1

Institut für Analytische Chemie, Justus-Liebig-Universität Gießen; Schubertstraße 60 – Geb. 16, 35392 Gießen 2 . Bundeskriminalamt, 65173 Wiesbaden In den Forensischen Wissenschaften kann die Frage nach dem Alter eines Dokumentes sehr häufig nicht eindeutig geklärt werden, weil das Altern eines Kugelschreibereintrages in hohem Maße von der (in der Regel unbekannten) ursprünglichen Zusammensetzung der verwendeten Kugelschreiberpaste und den Lagerungsbedingungen des Dokumentes abhängig ist. Generell bestehen Kugelschreiberpasten aus drei Hauptkomponenten: 1. Farbstoffe, 2. Lösungsmittel, 3. Harze und Bindemittel. Die drei Substanzklassen unterliegen unterschiedlichen Alterungsprozessen. Während Farbstoffe ausbleichen, d.h. vor allem photochemisch abgebaut werden, wird das Alterungsverhalten der Lösungsmittel durch den Verdampfungs- und Diffusionsprozess charakterisiert. Sehr wenig ist bis heute über das Alterungsverhalten der Harze und Bindemittel der Kugelschreiberpasten bekannt. Klassische Analysemethoden beruhen auf der Freisetzung von Monomereinheiten durch Pyrolyse oder Thermodesorption und anschließendem Nachweis durch die Kopplung mit GC/MS. Der Nachteil dieser Untersuchungsmethode besteht darin, dass eine Vielzahl von thermisch erzeugten Nebenprodukten aus dem untersuchten Material freigesetzt werden kann, was die Identifizierung der verwendeten Harze/Bindemittel erheblich erschwert. Vielversprechender als die genannten klassischen Analysemethoden erscheint die direkte Identifizierung der molekularen Komponenten und deren Alterungsprodukten entweder mit Hilfe der MALDIoder der ESI-Massenspektrometrie. Erste Ergebnisse zeigen, dass sich diese Substanzklassen sehr gut durch MALDI- und ESI-MS charakterisieren lassen. Die Massenspektren zeigen alle sehr charakteristische und substanzspezifische Verteilungsmuster, die in einigen Fällen auch Aussagen über die Primärstruktur der Referenzsubstanzen zulassen. In dem Beitrag soll sowohl auf die Untersuchungsergebnisse bzgl. des Alterungsverhaltens der Farbstoffe und Lösungsmittel, als auch auf erste Ergebnisse im Bereich der Harz/Bindemittel-Analytik eingegangen werden.

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Lecture V95 Real-time analysis of individual bioaerosol particles and single cells with bioaerosol mass spectrometry Matthias Frank1, James M. Birch1, Keith R. Coffee1, David P. Fergenson1, Maurice E. Pitesky1, Abneesh Srivastava1, Paul T. Steele1, Herb J. Tobias1, Vincent J. Riot1, Sue I. Martin1, Eric E. Gard1, Carlito Lebrilla2, Gregg A. Czerwieniec2, Scott C. Russel2, Erica L. McJimpsey.2 1

Lawrence Livermore National Laboratory, Livermore CA 94551, USA. 2 University of California Davis, Davis CA 95616, USA.

We are developing a single-particle bioaerosol mass spectrometry system that can rapidly analyze individual biological particles (e.g. spores or vegetative bacteria) that are sampled directly from air into a mass spectrometer. Mass spectra from both positive and negative ions created by matrix-free or matrix-assisted laser desorption and ionization are recorded simultaneously and can be analyzed and classified in real-time. Our present system is capable of discriminating, particle by particle, between bacterial spores and other biological and non-biological background materials using the mass fingerprints obtained from those particles. We will describe recent results including the comprehensive identification of critical marker peaks from spores by isotope labeling, the reduction of the shot-to-shot mass spectral variability by laser profile conditioning, and the demonstration of sub-attomole detection limits for peptide standards. We will also discuss the prospects for potential future applications in the detection of respiratory diseases and present first results from work on aerosolized Mycobacterium tuberculosis. This work was performed under the auspices of U.S. Department of Energy by Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48.

Literatur [1] "Instantaneous Reagentless Identification of Individual Bioaerosol Particles," D.P. Fergenson, M.E. Pitesky, H.J. Tobias, P.T. Steele, G.A. Czerwieniec, S.C. Russell, C.B. Lebrilla, J.M. Horn, K.R. Coffee, A. Srivastava, S.P. Pillai, M.P. Shih, H.L. Hall, A.J. Ramponi, J.T. Chang, R.G. Langlois, P.L. Estacio, R.T. Hadley, M. Frank and E.E. Gard, Anal. Chem. 76, 373-378 (2004). [2] "Laser Power Dependence of Mass Signatures from Individual Bacterial Spores in Bioaerosol Mass Spectrometry,” P.T. Steele, H.J. Tobias, D.P. Fergenson, M.E. Pitesky, J.M. Horn, G.A. Czerwieniec, S.C. Russell, C.B. Lebrilla, E.E. Gard, and M. Frank, Anal. Chem. 75, 5480-5487 (2003). [3] "Toward Understanding the Ionization of Biomarkers from Micrometer Particles by Bioaerosol Mass Spectrometry,” S.C. Russell, G.A. Czerwieniec, C.B. Lebrilla, H.J. Tobias, D.P. Fergenson, P.T. Steele, M.E. Pitesky, J.M. Horn, M. Frank, and E.E. Gard, J. Am. Soc. Mass Spectrom. 15, 900-909 (2004).

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Lecture V101 Stable Isotope-free Relative and Absolute Quantitation of Protein Phosphorylation Stoichiometry by Mass Spectrometry Hanno Steen, Judith A. Jebanathirajah, Michael Springer, Marc W. Kirschner Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA Qualitative and quantitative information are crucial to a detailed understanding of the function of the protein phosphorylation. Mass spectrometry, the method of choice for the qualitative, residue-resolved analysis of phosphorylation, has recently been introduced for the quantitation of protein phosphorylation as well. All methods described so far either require the elaborate and expensive use of stable isotopes to compare a limited number of samples or do not provide information about phosphorylation stoichiometries. Here we present mass spectrometric, stable isotope-free strategies which allow relative and absolute quantitation of protein phosphorylation stoichiometries. Using the methods developed: 1) We can account for run-to-run variations and variations in amounts of starting material using a robust normalization procedure; this procedure monitors the unmodified proteolytic peptides derived from the protein of interest and identifies those peptides which are suitable for normalization purposes. 2) We can determine changes in phosphorylation stoichiometry by monitoring the changes in the normalized ion currents of the phosphopeptide(s) of interest, i.e. relatively quantitate protein phosphorylation. 3) Absolute phosphorylation stoichiometry can be measured by monitoring the ion currents of a particular phosphopeptide and its unmodified cognate as the changes in the signal intensities of the phosphorylated and unphosphorylated form of a peptide are correlated. 4) We can extend the method such that it is applicable to multiply phosphorylated species; for this, one more sample with varying degrees of phosphorylation than number of phosphorylation sites is required to correct for the differences in the ionization/detection efficiencies of the phosphopeptide, its partially phosphorylated and its unphosphorylated cognate. 5) Species with ragged ends that result from incomplete proteolytic cleavages are amenable. These approaches were validated and subsequently applied to the quantitative phosphorylation analysis of proteins involved in transcription and MAPK signalling.

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Lecture V102 Anreicherung und massenspektrometrische Identifizierung phosphorylierter Peptide C. Sykora1, R. Hoffmann1, P. Hoffmann1 1

Universität Leipzig, Fakultät für Chemie und Mineralogie, BiotechnologischBiomedizinisches-Zentrum, Deutscher Platz 5, 04129 Leipzig, Deutschland

Die Phosphorylierung von Proteinen stellt einen wichtigen Regulationsmechanismus dar, der z.B. die Aktivität, Lokalisation oder die Interaktion mit anderen Proteinen beeinflusst. Bei der Identifizierung der Phosphorylierungsstellen bedarf es großer Sorgfalt, da schon während der Probenaufarbeitung Phosphorylierungsstellen aufgrund endogener Phosphataseaktivität verloren gehen können. Bei der Massenanalyse werden Phosphopeptide in Gemischen diskriminiert, da diese wegen ihrer negativen Ladung am Phosphat nur schlecht ionisiert werden1. Ziel dieser Arbeit war es, eine optimierte Analysemethode zur Identifizierung aller Phosphorylierungsstellen, insbesondere mehrfach phosphorylierter Sequenzen, eines Proteins/Peptids zu etablieren, basierend auf einer immobilisierten Metallionen Affinitätschromatographie (IMAC) Technik. Als entsprechende Systeme standen die kommerziell erhältlichen stationären Phasen von Perbio Science Deutschland GmbH (Phosphopeptide Isolation Kit, Ga3+ Ionen2,3) und Bruker Daltonics GmbH (Phosphopeptide Capturing Kit MB-IMAC Fe) zur Verfügung. Die Kits wurden im ersten Schritt an synthetischen Peptiden mit unterschiedlichem Phosphorylierungsgrad (nicht, einfach und zweifach) getestet. Anhand dieser Tests wurden die vom Hersteller beschriebenen Bedingungen optimiert. Die Peptide wurden im unteren pmol Bereich aufgetragen und mit den Kit-spezifischen Lösungen eluiert. Als reales Modell dienten tryptische β–Casein-, Ovalbumin- und BSA-Peptidmischungen, die ebenfalls einen unterschiedlichen Phosphorylierungsgrad aufwiesen. Die zurückgehaltenen Peptide wurden anschließend mit MALDI-TOF/TOFMS charakterisiert.

Literatur: [1] Raska, C.S.; Parker, C.E.; Dominski, Z.; Marzluff, W.F.; Glish, G.L.; Pope, R.M.; Brochers, C.H. Anal. Chem. 2002, 74, 3429-3433 [2] Posewitz, M.C.; Tempst, P.; Anal. Chem. 1999, 71, 2883-2892 [3] Hoffmann, P.; Olayioye, M.A.; Moritz, R.L.; Lindeman, G.J.; Visvader, J.E.; Simpson, R.J.; Kemp, B.E. Electrophoresis, accepted

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Lecture V103 Phosphorylation site mapping by a multi-protease approach with highly specific phosphopeptide enrichment Andreas Schlosser1, Jens Vanselow2, and Achim Kramer2 Institut für Medizinische Immunologie, Charité, Universitätsmedizin Berlin 1 Mass Spectrometry Lab, 2 AG Chronobiologie Protein phosphorylation is one of the most ubiquitous cellular regulation mechanisms involved in a large variety of cellular processes. For the identification of phosphorylation sites several mass spectrometry-based methods have been established, the majority of which utilises either phosphopeptide specific scan techniques, such as neutral loss or precursor ion scan, or methods for phosphopeptide specific enrichment, most commonly immobilised metal affinity chromatography (IMAC). Several years ago Nakamura et al. discovered the chemo-affinity of titania (TiO2) for organic phosphates [1]. More recently, titania was used for the selective enrichment of phosphopeptides [2,3]. We have further optimised this procedure and established a multiprotease approach with highly specific phosphopeptide enrichment. The principle of this approach is outlined in Figure 1. trypsin

elastase complex peptide mix

phosphoprotein

selective enrichment with TiO2

phosphopeptides

proteinase K

thermolysin

Figure 1: The multi-protease approach. Phosphoproteins are digested in parallel with four different proteases. The four samples are pooled, and phosphopeptides are selectively enriched by a TiO 2-column from this highly complex peptide mixture. Phosphopeptides are analysed with ultra-low flow nanoLC-MS/MS.

Self-made nano columns filled with 5µm-TiO2 particles (Titansphere, GLSciences, Japan) are used for the selective phosphopeptide enrichment. The eluates from these affinitycolumns are analysed with ultra-low flow nanoLC-MS/MS (25 nL/min flow rate). With optimised washing conditions TiO2 has proved to bind phosphopeptides highly specific, whereat phosphopeptide recoveries seem to be nearly quantitative even in the low femtomole range. The use of four different proteases results in much more complete phosphorylation site maps compared to trypsin digest alone. Literature [1] Matsuda H, Nakamura H, Nakajima T Anal. Sci. 1990, 6, 911. [2] Sano A, Nakamura H Anal. Sci. 2004, 20, 565. [3] Pinske MWH, Uitto PM, Hilhorst MJ, Ooms B, Heck AJR Anal. Chem. 2004, 76, 3935.

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Lecture V104 Evaluierung von LC-ICP-MS und LA-ICP-MS zur Bestimmung des Protein- oder Proteom-Phosphorylierungsgrades Ralf Krüger, Wolf D. Lehmann Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Zentrale Spektroskopie Die reversible Phosphorylierung von Proteinen ist ein zentraler Mechanismus der zellulären Kommunikation. Die Signaltransduktion erfolgt dabei über eine Veränderung des Phosphorylierungsgrades. Die Bestimmung des prozentualen Anteils an phosphorylierten Isoformen kann somit wertvolle Aussagen über den funktionellen Zustand von Phosphoproteinen und deren Interaktionspartnern liefern. ICP-MS eignet sich gut zur Bestimmung des Protein-Phosphorylierungsgrades via simultaner Phosphor- und Schwefel-Detektion [1]. Bei bekanntem Gehalt an schwefelhaltigen Aminosäuren ist die 34S- oder 32S-Intensität ein Maß für die Proteinmenge, während der Grad der Phosphorylierung über die Intensität von 31P errechnet werden kann (Abb. 1). Dazu ist keine absolute Quantifizierung, sondern nur die Bestimmung der relativen Signalempfindlichkeit dieser Isotope für die jeweilige Matrix und Methode notwendig. Phosphor / Protein: 1.45 mol / mol

intensity [cps]

400000

300000

31

P S

34 200000

100000

0 0

500

1000

1500

2000

2500

Abb. 1: rp-LC-ICP-MS Chromatogramm von intaktem Ovalbumin (G. 31 34 gallus) mit P- und S-Detektion zur Analyse des Phosphorylierungsgrades (Literaturwert: 1.73 mol / mol [2]).

time [sec]

Eine Kontamination mit phosphor- oder schwefelhaltigen Verbindungen (z.B. Phospholipide, Nucleotide) muß ausgeschlossen sein. Daher ist eine Aufreinigung / Trennung der Proteine durch Gelelektrophorese oder HPLC unerläßlich, wobei die Kombination mehrerer Methoden auch eine Analyse komplexerer Mischungen ermöglicht. Drei Ansätze wurden anhand von Referenz-Phosphoproteinen untersucht:  LC-ICP-MS der intakten Proteine  SDS-PAGE, enzymatischer Verdau und LC-ICP-MS der Peptide  SDS-PAGE / Western Blot und Laserablation(LA)-ICP-MS der intakten Proteine Alle Varianten erlauben die schnelle Bestimmung des Protein-Phosphorylierungsgrades auf etwa ± 20 %. Die Methode läßt sich auf Proteinmischungen und Proteome erweitern. Vergleichende Untersuchungen von cytosolischen Proteinpräzipitaten (Sub-Proteom) von verschiedenen Zellstadien- und Organismen werden vorgestellt. Literatur [1] Wind, Wesch, Lehmann, Anal. Chem. 73, 2001, 3006 [2] Ekman, Jäger, Anal. Biochem. 214, 1993, 138

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Lecture V111 Mapping Protein Interfaces by Affinity Cross-Linking Combined with MALDI-TOF and ESI-FTICR Mass Spectrometry Andrea Sinz, Stefan Kalkhof and Christian Ihling Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, D-04103 Leipzig, Germany Chemical cross-linking of protein complexes, a conceptually simple approach, has gained renewed interest in combination with mass spectrometric analysis of the reaction products [1]. This approach allows a rapid mapping of protein interfaces, which is crucial for understanding protein/protein interactions. The identification of cross-linking products from the complex mixtures created after the cross-linking reaction however remains a daunting task. In order to facilitate the identification of cross-linking products, we explore the use of the commercially available biotinylated cross-linking reagent sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3'dithiopropionate). This trifunctional cross-linker possesses one amine-reactive and one photo-reactive site and additionally allows an affinity-based enrichment of cross-linker containing species [2]. As model system, we chose the Ca2+-dependent complex between calmodulin and its target peptide M13, which had already been studied by chemical crosslinking and ESI-FTICR (electrospray ionization Fourier transform ion cyclotron resonance) mass spectrometry [3]. The peptide M13 comprises 26 amino acids and represents a part of the C-terminal sequence of the skeletal muscle myosin light chain kinase. After the cross-linking reaction, MALDI-TOFMS and one-dimensional gel electrophoresis (SDS-PAGE) were employed to check the extent of cross-linking product formation. The cross-linking reaction mixtures were subjected to tryptic in-solution digestion. Biotinylated peptides, e.g., peptides that had been modified by the cross-linker as well as cross-linked peptides, were enriched on monomeric avidin beads after a number of washing steps had been performed. Peptide mixtures were analyzed by MALDI-TOFMS and nano-HPLC/nano-ESI-FTICRMS. We demonstrate that an enrichment of cross-linker containing species allows a more efficient identification of interacting amino acid sequences in protein complexes. This strategy is expected to be especially beneficial for investigating large protein assemblies. [1] Sinz, A. (2003) J.Mass Spectrom. 38, 1225-1237. [2] Hurst, G.B., Lankford, T.K., Kennel, S.J. (2004) J. Am. Soc. Mass Spectrom. 15, 832-839. [3] Kalkhof, S., Ihling, C., Mechtler, K., Sinz, A. Anal. Chem., Web Release Dec. 7, 2004.

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Lecture V112 Characterization of peptide-protein and protein-protein interactions using photoaffinity labeling and mass spectrometry Olaf Jahn 1,2,3, Harald Junge 2, Klaus Eckart 1, Joachim Spiess 1, and Nils Brose 2 1

Molecular Neuroendocrinology, 2 Molecular Neurobiology, 3 Proteomics Group, MaxPlanck-Institute for Experimental Medicine; Hermann-Rein-Str. 3, 37075 Göttingen

Interactions of macromolecules such as peptides and proteins can be biochemically investigated by photoaffinity labeling (PAL) and subsequent mass spectrometric characterization of the resulting photoadducts. Depending on the analytical strategy applied, a PAL experiment can provide different levels of information, ranging from the identification of binding partners to the structural characterization of ligand-binding sites. In this presentation, two different macromolecular interactions will be discussed. First, the characterization of ligand-binding sites will be demonstrated on the basis of photoadducts formed by analogs of the neuropeptide corticotropin-releasing factor (CRF) and its binding protein (CRFBP) [1]. By using LC-MS and MS/MS, these 41 kDa photoadducts were analyzed at the level of the single amino acids involved and, thereby the ligand-binding site of CRFBP was mapped to amino acids 23-36 in the N-terminal part of the protein. In future, these data will be helpful to facilitate structure-based design of CRFBP inhibitors proposed to have therapeutical potential for the treatment of cognitive deficits. The second example originates from a recent study on the interaction of the ubiquitous calcium sensor protein calmodulin (CaM) with Munc13 proteins, which are known to function as essential regulators of synaptic vesicle priming and synaptic efficacy. With the help of photoreactive peptides mimicking a putative CaM recognition sequence of 200 kDa Munc13, binding of CaM to Munc13 at 1:1 stoichiometry was shown by LC-MS, and thereby an evolutionary conserved CaM binding site in Munc13 was established. In the framework of this study, the CaM/Munc13 complex was identified as the molecular correlate for the known effects of residual cytosolic calcium on synaptic plasticity [2]. Literatur [1] Jahn, O.; Eckart, K.; Brauns O.; Tezval, H.; Spiess, J. The binding protein of corticotropin-releasing factor: Ligand-binding site and subunit structure. Proc. Natl. Acad. Sci. USA 2002; 99:12055-12060. [2] Junge, H. J.; Rhee, J.-S.; Jahn, O.; Varoqueaux, F.; Spiess, J.; Waxham, M. N.; Rosenmund, C.; Brose, N. Calmodulin and Munc13 form a Ca2+ sensor/effector complex that controls short-term synaptic plasticity. Cell 2004; 118:389-401.

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Lecture V113 Potential of functional proteomics: An epilepsy gene product slows K+ currents by assembly with Kv1.1- polyprotein complexes Uwe Schulte1, Jörg Oliver Thumfart2, Nikolaj Klöcker2, Claudia Sailer3, Wolfgang Bildl2, Martin Biniossek4, Thomas Deller5, Silke Eble2, Karen Abbass1, Tanja Wangler1, Hans-Günther Knaus3, Bernd Fakler2 1

Complexio GmbH, Hermann-Herder-Str. 7, 79104 Freiburg, Germany. Institute of Physiology, University of Freiburg, Hermann-Herder-Str. 7, 79104 Freiburg, Germany. 3 Institute of Biochemical Pharmacology, University of Innsbruck, Peter-Mayr-Str. 1, A-6020 Innsbruck, Austria. 4 Institute of Molecular Medicine and Cell Research, University of Freiburg, StefanMeier-Str. 17, 79104 Freiburg, Germany. 5 Department of Clinical Neuroanatomy, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany 2

Our functional proteomics attempt was the identification of all or at least a majority of interacting proteins of the Kv1.1 ion channel. Therefore we performed co-immunoprecipitations (CoIP) of this K+ ion channel and separated the precipitation products with SDS-PAGE. Kv1.1 specific bands were excised, in-gel digested and identified via nanoflow liquid chromatography mass spectrometry (nLC-MS) with subsequent Mascotsearches. In the list of all identified proteins from this experiment, we found all proteins in the literature described as likely interaction partners for Kv1.1, and some candidates with no described function related to this ion channel. With the most interesting protein candidate in this list we performed a reverse CoIP and we were able to identify Kv1.1 by mass spectrometry in the opposite direction, too. Idiopathic epilepsies are predominantly caused by genetic disorders with all but one of the identified disease genes coding for voltage- or transmitter gated ion channels. The exception to the rule is LGI1 (leucine-rich glioma inactivated gene 1) originally cloned from the translocation breakpoint of a glioma cell line and found to be responsible for auto-somal dominant form of lateral temporal lobe epilepsy (ADTLE). The normal cellular function and distribution within the cell are not finally known. Here we show that LGI1 is part of the protein complexes associated with the voltagegated K+ channel Kv1.1. Immunocytochemistry showed a distinct distribution of this protein with a most prominent expression in the hippocampal perforant path where it colocalizes with the Kv channel subunits Kv1.1, Kv1.4 and Kvbeta 1. Functional expression of these subunits in Xenopus oocytes showed that LGI1 considerably slows the rapid inactivation gating of the resulting heteromeric channels without significant changes in channel activation. In contrast, C-terminal deletions of disease gene product identified in patients with ADTLE failed to exert any functional effect.

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Lecture V114 Quantiative Analysis of the Phosphotransferase System in Escherichia Coli by Metabolic Labeling and MALDI Mass Spectromery 1 G. Franke, 1 Kai Reidegeld, 2 M. Siemann, 2 M. Reuss, 1 H.E. Meyer, 1 B. Warscheid; 1 Medical Proteom-Center, University of Bochum, Bochum, Germany; Universitätsstr. 150, 44801 Bochum, Phone: + 49 234 32 29263, Fax: + 49 234 32 14554 2 Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany; Allmandring 31, 70569 Stuttgart, Phone: +49 711 685 5161, Fax: +49 711 685 5164 Modeling of metabolic pathways is widely used to provide deeper insight into intracellular carbon fluxes in microorganisms. To develop more accurate models and improve bacterial production processes, detailed information on protein expression profiles is needed. In this work, we have focused on the phototransferase system (PTS) triggering carbohydrate transport, regulation and chemotaxis in bacteria.To obtain differential expression data on the PTS subproteome in E. coli at different growth rates, a metabolic labeling strategy has been applied enabling relative quantitative analysis by mass spectrometry. Additionally, a customized software has been developed and successfully employed for quantitative analysis of mass spectral data. E. coli DSM 10122 was cultivated under fed batch conditions with medium containing 14Nammonium sulfate and samples were taken at growth rates of 0.036, 0.072, 0.09, and 0.124. For subproteome expression profiling, E. coli DSM 10122 was grown under comparable conditions with 15N-ammonium sulfate as single nitrogen source and harvested at a growth rate of 0.09. Each of the bacterial cell samples labeled with the 'light' isotopes were directly mixed with the sample aliquots containing the 'heavy' isotopes. After cell lysis, cytosolic protein fractions were separated by two-dimensional gel electrophoresis (2-D PAGE). Protein spots were picked, digested with trypsin, and peptides generated were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI/TOFMS) combined with bioinformatics for protein identification and relative quantitation. Up- or down-regulation of proteins at defined growth rates was determined by comparative analysis of about 3 - 5 characteristic protonated peptide pairs observed in the MALDI mass spectra, and statistical analysis was further supported by up to 6 individual 2D gel preparations. Additionally, densitometric analysis of Colloidal Coomassie stained gels was performed with the software ProteomWeaver®. The quantitative information on protein spots achieved were compared to quantitative results from metabolic labeling experiments with stable isotopes and subsequent MS analysis.

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Lecture V121 Die Fragmentierung von Even Electron Ionen Karsten Levsen1, Hans-Martin Schiebel², Alfred Preiß1 Manfred Elend1, Herbert Thiele³ und Arnd Ingendoh³, 1

Fraunhofer Institut für Toxikologie und Experimentelle Medizin; Hannover, ²Technische Universität, Braunschweig; ³Bruker Daltonik, Bremen

Moderne Ionisierungsmethoden, wie ESI und APCI, die heute speziell in der Kopplung mit der HPLC in der Arznei- und Pflanzenschutzmittelforschung, aber auch bei der Untersuchung von Naturstoffen und Biopolymeren eingesetzt werden, liefern fast ausschließlich Quasimolekülionen mit gerader Elektronenzahl (Even Electron Ionen), d.h. insbesondere [M+H]+ und [M-H]- Ionen, während bei der klassischen Elektronenstoßionisation (EI) Molekülionen mit ungerader Elektronenzahl (Odd Electron Ionen) entstehen. Während die Zerfallsregeln der Odd Electron Ionen in der EI wohl bekannt und in vielen Publikationen und Lehrbüchern dokumentiert sind, ist der Zerfall der Even Electron Ionen bisher nicht systematisch untersucht worden. Um diese Lücke zu schließen, haben wir den stoßinduzierten Zerfall von ca. 150 bevorzugt aromatische Verbindungen mit ein bis drei verschiedenen funktionellen Gruppen in einer Ionenfalle untersucht, wobei die Ionisierung unter ESI oder APCI Bedingungen erfolgte. Beim Zerfall dieser niederenergetischen Ionen spielen überwiegend energetische und weniger kinetische Faktoren eine Rolle. Die Fragmentierung der [M+H]+ und [M-H]- Ionen unterscheidet sich z. T. signifikant von dem in der EI beobachteten Zerfall, wobei der Verlust von stabilen kleinen, thermodynamisch günstigen Molekülen, wie H2O, NH3, CO, CO2, HCN und SO2 unter Bildung von Even Electron Ionen im Einklang mit der „Even Electron Regel“ erfolgt. Es werden jedoch, insbesondere bei aromatischen Verbindungen, zahlreiche Ausnahmen von dieser Regel beobachtet, die sich, wie in Beispielen gezeigt wird, anhand der thermodynamischen Daten erklären lassen. Die (stoßinduzierten) Fragmentionen von [M+H]+ und [M-H]- Ionen können bei der Identifizierung unbekannter Verbindungen zum Erkennen funktioneller Gruppen genutzt werden. Allerdings sind die dabei auftretenden Neutralteilverluste nur zum Teil charakteristisch und spezifisch für eine funktionelle Gruppe. So lassen sich insbesondere Carboxyl-, Sulfon- und Nitrogruppen, nicht jedoch aromatisch gebundene Hydroxyl- und Aminogruppe durch einen charakteristischen Neutralteilverlust identifizieren. Es wird gezeigt, dass zur eindeutigen Charakterisierung zusätzliche Hochauflösungsdaten dringend erforderlich sind.

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Lecture V122 The Proton Affinity of Liquid Crystal Model Compounds Daniel Kühne, Karl-Peter Wanczek University of Bremen, Institute for Inorganic and Physical Chemistry, Haferwende 12, 28357 Bremen Thermotropic liquid crystals are a very important special product of chemical industry. For modern display technology (TFT-displays for notebook and desktop computers, cell phones and PDAs as well as LCD-TVs), liquid crystal compounds are indispensable. The image quality of active matrix (AM) displays strongly depends on the “reliability” of the liquid crystal compounds used. Liquid crystals with high ion affinities will solvate impurity ions from peripheral display materials. These ions will reduce the specific resistivity and therefore cause display flickering and image sticking.[1] A modified Bruker prototype FT ICR Spectrometer with 7 Tesla superconducting magnet and a closed cylindrical ICR cell was employed for proton affinity determination. Liquid crystal compounds were introduced into the vacuum system with a special solid sample inlet system, mounted on top of the ICR cell. Volatile reference compounds were introduced by a standard gas inlet system. Proton affinities were determined by equilibrium method. Computational calculations were carried out using the GAUSSIAN software package.[2] The proton affinity of 10 liquid crystal compounds and two liquid crystal building blocks were determined. The compounds were chosen in a manner that the influence of different substitution on proton affinity and structure of the protonated molecule may be analysed. The investigated compounds are: 1-(Pentafluoro-6-sulfanyl)-4-(4'-propylcyclohexyl)-benzene 2-(4-(Pentafluoro-6-sulfanyl)-phenyl)-5-propyl-[1,3]-dioxane 2-(4-(Pentafluoro-6-sulfanyl)-phenyl)-5-propyl-[1,3]-dithiane 4-(pentafluoro-6-sulfanyl)-phenyl-4-propylcyclohexanecarboxylate 5-Ethyl-2-(4-(pentafluoro-6-sulfanyl)-phenyl)-tetrahydropyran 1,2,3-Trifluoro-5-(4'-(4''-propyl-cyclohexyl)-cyclohexyl)-benzene 1,2,3-Trifluoro-5-(2'-(4'-(4''-propyl-cyclohexyl)-ethyl)-cyclohexyl)-benzene 6) 1,2,3-Trifluoro-5-(2',6'-dioxo-4'-(4''-propyl-cyclohexyl)-cyclohexyl)-benzene 7) 1,2,3-Trifluoro-5-(difluoromethoxy-4'-(4''-propyl-cyclohexyl)-cyclohexyl)-benzene 8) 1,2,3-Trifluoro-5-(difluoromethoxy-(2',6'-difluoro-4'-(4''-propylphenyl)-phenyl)-benzene 9) S,S-Diphenyl-N-(2,3,4,5,6-pentafluorophenyl)-sulfimide 10) S,S-Diphenyl-N-(2,3,4,5,6-pentafluorophenyl)-sulfoximide 1) 2) 3) 4) 5)

The site of protonation differs with substitution and was analysed with computational calculations. Quantitative results and structures will be presented in detail. The liquid crystal compounds 1), 5), 9) and 10) show HF-loss after protonation. Theoretical calculations confirm this observation. This results and – where neccessary – the corresponding reaction mechanisms will also be presented. [1] P. Kirsch, M. Bremer, Angew. Chem. Int. Ed., 39 (2000), 4216 [2] Gaussian 03, Revision C.02, J.A. Pople et al., Gaussian, Inc., Wallingford CT, 2004.

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Lecture V123 Monitoring nucleation reactions by coupling different reactors to an ESI Mass Spectrometer: about silicate species and zeolithe precursors Pelster S., Schüth F., Schrader W. Max-Planck Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr Condensation of monomers leads to oligomeric units that conglomerate further more in different nucleation steps to bigger species. When the crystallization point is reached the growth of the crysal can be observed macroscopically. The knowledge of these processes involved in the formation of a solid is rare and limited and often restricts to empirical findings. Better understanding of solid state formation could help to design more efficient heterogeneous catalysts like zeolithes. But while it is possible to analyze the precursors and even the solids using a number of different analytical methods, the observation of the nucleation process is still problematic. We have developed a procedure to analyze inorganic aqueous silicates with different mass spectrometers utilizing electrospray-ionization [1, 2]. Although the working conditions are beyond standard conditions due to high salt content of the samples in the percent range, it was possible to characterize a number of different species and to address questions such as charge and degree of hydrolysis of the species present in solution. To be able to get further insight into the silicate chemistry and especially the nucleation process we have coupled a reactor to a mass spectrometer. This combination allows a detailed and time-resolved study of the behavior of silicates in solution with regard to their environment. To study different reaction conditions we used four silicate reactants such as alkoxysilanes and three counter-ions in regard to the formation of different silicate oligomers. The nature of the alkoxysilanes affects basically the reaction rate caused by slower or faster hydrolysis. Thus, choosing the adequate alkoxy lenght enables detailed observation of every interesting reaction step in the nuceation process. The counter-ions are composed from tetraalkylammoniumhydroxyd which plays a major role in the formation of the silicate species. Adding specific crosslinkers, thermal treatment and adjusting the pHvalue leads to conditions closer to the crystallization point, where larger species are formed that are supposed to be precursors for the zeolithe synthesis [3, 4]. Literatur [1] Bussian P., Sobott F., Brutschy B., Schrader W., Schüth F., Angew. Chemie Int. Ed., (2000) 39 (21), 39013905; Angew. Chemie, (2000) 112 (21), 4065 [2] Pelster S., Schrader W., Schüth F., in preparation [3] Agren P., Thomson S., Ilhan Y., Zibrowius B., Schmidt W., Schüth F., Stud.Surf.Sci.Catal., (2002) 142, 159-166 [4] Ravishankar R., Kirschhock C.E.A., Schoeman B.J., Devos D., Grobet P.J., Jacobs P.A., Martens J.A., Proceedings of the 12th Zeolithe Conference MRS, Pennsylvania, (1999), 1825-1832

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Lecture V124 Probing the Mechanism of Classical and the Supramolecular CoCatalyzed Baylis-Hillman Reactions via ESI(+)-MS and MS/MS L. S. Santos,* C. H. Pavam, C. S. Consorti, B. A. S. Neto, F. Coelho, J. Dupont and M. N. Eberlin *University of Oldenburg, Department of Chemistry, D-26111 Oldenburg, Germany The Baylis–Hillman reaction has recently emerged as extremely versatile method for the synthesis of highly complex systems. The reaction leads to the straightforward formation of new  C-C bonds in a single step, and yields functionalized molecules (-methylene-hydroxy derivatives), which are then conveniently manipulated as key synthons in the synthesis of a variety of natural and nonnatural products. In the current work, the proposed intermediates for the catalytic cycle of the Baylis–Hillman reaction (Scheme 1) have been successfully intercepted and structurally characterized for the first time using electrospray ionization with mass and tandem mass spectrometry. Strong evidence for the currently accepted mechanism has been collected, thus confirming the proposals initially made by Isaacs and Hill.1 Herein, attempts to intercept key cationic intermediates of Baylis–Hillman reactions performed in ionic liquids have been obtained. Several H-bonded cationic species formed by ion pairing of the aldehyde and zwiterionic intermediates with ionic liquid cations and anions have been transferred from solution to the gas-phase, detected and characterized. These supramolecular species indicate therefore that ionic liquids improve overall reaction rates by activating the aldehyde toward nucleofilic attack and by stabilizing the zwitterionic species that acts as the main Baylis-Hillman intermediates.2 +

+

N N + OH

N N+ H

BMI.Y

OCH3 [5 + H]+ m/z 199

[3 + H]+ m/z 113

BMI+ Y+

O N

O H

N

O

S 2

S

H

1 +

+

8 m/z 252

BMI.Y

N + N OH

N N+ O OCH3

OCH3

N

N

N

HO 7

HO

S

OH BMI+

BMI.Y

S

Y-

OCH3 N S

11a+, Y- = BF4-, m/z 538

m/z 312

11b+, Y- = PF6-, m/z 596

CO2CH3

N

4 Baylis-Hillman Adduct

12 m/z 338

BMI+

S [7 + H]+

S +

N N + OH

HO

OH CO2CH3

N

+

BMI.Y

OCH3

S

6

OCH3

BMI

10c+, Y- = CF3CO2-, m/z 511

N N+ O

5

O

OCH3

10b+, Y- = PF6-, m/z 543

9c+, Y- = CF3CO2-, m/z 451

OCH3

OH

-

10a+, Y- = BF4-, m/z 485

-

H

N + N Y

H3CO

9b+, Y- = PF6-, m/z 483

N N+ O

N N DABCO 3

-

N N+

OH

+

OCH3

9a , Y = BF4 , m/z 425

H+

H+

N N + OH

+ N N

H H+

11c+, Y- = CF3CO2-, m/z 564 H+

O

O

N

OCH3 S [4 + H]+ m/z 200

DABCO 3

Scheme 1: Baylis-Hillman reaction of methyl acrylate (1) and thiazolecarboxaldehyde 2 cocatalyzed by both DABCO (3) and ionic liquids BMI.Y (Y-=BF4-, PF6-, CF3CO2-) showing both the protonated and supramolecular H-bonded cationic species gently transferred from solution to the gas phase by ESI, detected and structurally characterized by MS and MS/MS analysis, with their respective m/z ratios. Hill, J. S., Isaacs, N. S. J. Phys. Org. Chem. 1990, 3, 285. 2) Santos, L. S., Pavam, C. H., Almeida, W. P., Coelho, F., Eberlin, M. N Angew. Chem. Int. Ed. 2004, 43, 4330.

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Lecture V125 Mit Tyramin auf Spurensuche im Schlafmohn Jürgen Schmidt1, Chotima Poeaknapo2, Christine Kuhnt1, Toni M. Kutchan1 und Meinhart H. Zenk2 1

Leibniz-Institut für Pflanzenbiochemie, Weinberg 3, D-06120 Halle/Saale. 2Biozentrum der Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, D-06120 Halle/Saale

Tyramin steht am Anfang eines Biosynthesewegs, der in Papaveraceen zu unterschiedlichen Alkaloidtypen führt. Dazu gehören u. a. pharmazeutisch wichtige Alkaloide mit Benzylisochinolin-, Protoberberin- und Morphinan-Grundgerüst [1]. In der Biosynthese von Papaver-Alkaloiden kommt dem (S)-Reticulin eine Schlüsselrolle zu, da über dieses Benzyltetrahydroisochinolinalkaloid verschiedene Alkaloidtypen gebildet werden [2, 3]. Fütterungsexperimente mit 13C6-Tyramin an Keimlinge von Papaver somniferum und anschließende massenspektrometrische Analysen mittels ESI-FT-ICR-MS und LC/ESIMS/MS erlauben es, im Sinne einer „Momentaufnahme“ eine Reihe von Alkaloiden aufzuspüren, die aus diesen Präkursoren gebildet werden. Der vorliegende Beitrag diskutiert die Möglichkeiten einer Kombination von tandemmassenspektrometrischen Methoden mit einem Triple-Quadrupole-Instrument und dem Potenzial der exakten Massenbestimmung mittels FT-ICR-MS für die Evaluation von Fütterungsexperimenten am Beispiel von Papaver-Alkaloiden. H3CO N HO

CH3

H

OH

OCH3

(S)-Reticulin Literatur [1] T. M. Kutchan, Molecular genetics of plant alkaloid biosynthesis. In Cordell, G. (Ed.), The Alkaloids 50, 257-316 (1998) [2] W.-M. Chou und T. M. Kutchan, Enzymatic oxidations in the biosynthesis of complex alkaloids. Plant J. 15, 289-300 (1998) [3] K. Hirata, C. Poeaknapo, J. Schmidt und M. H. Zenk, 1,2-Dehydroreticuline synthase, the branch point enzyme opening the morphinan biosynthetic pathway. Phytochemistry 65, 1039-1046 (2004)

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Poster P1 XACT: Development of a New Method for Studying Protein-Protein Interactions Ahrends, R1., Kirsch, D.2, Spengler, B.2 and Friedhoff, P.1 1 2

Institute of Biochemistry, Justus-Liebig-Universitaet Giessen; Institute of Analytical Chemistry, Justus-Liebig-Universitaet Giessen

A fast method is presented to analyze protein-protein (e.g. antigen-antibody) interaction sites by a combination of photoactivatable cross-linking, affinity purification and mass spectrometry, XACT (X-linking-Affinity purification, chemical Coding Technology). The bacterial mismatch repair (MMR) system of E. coli serves as a model to establish these new methods. This highly conserved system is present in most organisms and is mainly involved in correcting replication errors, thereby enhancing the fidelity of DNA replication by a factor of up to 1000[1]. In this study, the MMR proteins are selectively modified with trifunctional cross-linkers containing a biotin and benzophenone moiety at single cysteine residues introduced by site-directed mutagenesis. Upon complex formation with partner proteins (e.g. MutH or MutS), photocross-linking is performed[2]. These complexes are then digested with proteases and the cross-linked peptide carrying the biotin is selectively isolated from other peptides using magnetic beads[3] followed by chemical coding. Read-out is performed using MALDI MS, with subsequent identification. This approach accelerates the detection of protein interaction sites needed as experimental constraints for successful protein-protein docking algorithms to build molecular models of protein complexes (e.g. the MMR system or targets of the immune response). This work was supported by the Deutsche Forschungsgemeinschaft FR 1495/3-1 and the BMBF, Germany, Grant No.0312834A. Literature [1.] P. Modrich and R. Lahue: Missmatch repair in replication fidelity, genetic recombination and cancer biology, Annu. Rev. Biochem., 1986, 65: 101-133 [2.] Giron-Monzon L, Manelyte L, Ahrends R, Kirsch D, Spengler B, Friedhoff P: Mapping protein-protein interactions between MutL and MutH by cross-linking, J Biol Chem, 2004, 279: 49338-45. [3.]Girault, S., G. Chassaing, et al. : Coupling of MALDI-TOF mass analysis to the separation of biotinylated peptides by magnetic streptavidin beads, 1996, Anal Chem, 68: 2122-6.

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Poster P2 Ungewöhnliche Reaktionen von Indolizinen mit Chloroform in methanolischer Lösung unter Lichteinfluss M. Bartoszek1, H. Sonnenschein2 1

Institut für Angewandte Chemie Berlin-Adlershof, Richard-Willstätter-Str. 12, 12489 Berlin. 2 Angewandte Synthesechemie Adlershof, Richard-Willstätter-Str. 12, 12489 Berlin. Indolizine sind Redoxsysteme deren dimere Vertreter b) und c) als Redoxschalter genutzt werden können. Die Reversibilität des Redoxvorganges und die Lichtbeständigkeit sind dabei von der Art und Position des Substituenten abhängig.1,2. Bei der analytischen Charakterisierung einiger Vertreter dieses Verbindungstyps traten ungewöhnliche Peaks in den ESI-Spektren auf. Neben den Peaks für Dimere bei Gegenwart von Chloroform in der methanolischen Lösung fanden sich auch Spezies mit größeren Massen, die auf eine Anlagerung von Lösungsmittelmolekülen schließen ließen. Das charakteristische Molekül mit einer Masse von [2M+11]+ ist jedoch nur durch eine komplexe Reaktion mit CHCl3 zu erklären, nicht jedoch mit einfacher Anlagerung von Lösungsmittelmolekülen. Ph

Ph

Ph

Ph

Ph

N

N

N

R

R

R

N

N O

O O

a)

b)

O

O O

c)

R=H, Me, C2H4OH Anhand des Vergleichs der Reaktion von Indolizinen a), Biindolizinen b) und einem cyclischen Biindolizin c) konnte eine Vorstellung zum Mechanismus der Reaktion entwickelt werden. Die beschriebene Dimerisierungsreaktion unter Einbeziehung eines CHCl3-Moleküls wird nur unter Lichteinfluss beobachtet (normale Raumbeleuchtung). Deren zeitlicher Verlauf lässt sich gut verfolgen. Literatur [1] S. Hünig, H.Linhardt, Justus Liebiegs Ann. Chem. (1976) 317 [2] S. Hünig, H. Sonnenschein, J. Prakt. Chem. 336 (1994) 38-42

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Poster P3 Fast monitoring of the therapeutic effect of antibiotics in exhaled air by ion mobility spectrometry J.I. Baumbach 1, W. Vautz1, V. Ruzsanyi 1, L. Freitag 2 1

ISAS – Institut for Analytical Sciences, Bunsen-Kirchhoff-Str. 11, 44139 Dortmund 2 Lung Hospital Hemer, Theo-Funccius-Str. 1, 58675 Hemer.

Metabolites detectable in human exhaled air are correlated directly to different kinds of diseases, sometimes also to bacteria and molds. In the present paper, an ion mobility spectrometer (IMS) coupled to a multi-capillary-column (MCC) as pre-separation unit is used to identify and quantify volatile metabolites occurring in human breath down to the ng/L- and pg/L-range of analytes. In addition, the effect of drugs on the signal pattern obtained in IMS-chromatograms is related to a decrease of bacterial metabolites directly. The effect of drug delivery on patients showing Angina lateralis is presented exemplarly to show the potential of the method developed in the field of detection of pathways, effective dosage and dicision of effective time intervals to deliver pharmaceuticals. Case studies for Theophyllin used on chronic obstructive pulmonary disease (COPD), Axocillin used on Anginal lateralis and Orfiril are presented and discussed. The main advantage of IMS on direct analysis procedures and total analysis time less than 10 minutes show the potential for clinical applications. The aim of the studies is to introduce the investigation of metabolites in human breath as method for early recognition of selected diseases and monitoring of the effectivity of drug delivery on the basis of ion mobility spectrometry data.

110

Poster P4 Statistical approaches for the analysis of lung diseases by means of ion mobility spectrometry J.I. Baumbach 1, S. Bader 2, W. Urfer 2 1

ISAS – Institut for Analytical Sciences, Bunsen-Kirchhoff-Str. 11, 44139 Dortmund 2 Department of Statistics of the University of Dortmund, Vogelpothsweg 87, 44227 Dortmund .

Different lung diseases are related to different concentration profiles of volatile metabolites detectable in human breath by ion mobility spectrometry. Acetone is a marker for diabetes, nitric acid for asthma, ammonia for hepatitis and alkanes and the ratio of different benzene derivates for lung diseases. These conclusions were obtained by empirical investigation of metabolites in human breath measured by ion mobility spectrometry coupled to multicapillary columns within less than 10 minutes total analysis time at ambient pressure. Therefore, the enhanced aim is to approve these results by using statistical approaches for the processing of ion mobility spectrometry data and appreciate their validity by giving an estimation of the magnitude of the error rate. The ion mobility spectrometry data are used as input and transformed by baseline correction, reduced mobility scale, adjustment of retention time to temperature, class formation, peakfinder, cluster analysis and boundingbox-algorithms, definition of centroids for different detection regions, selection of variables, Wilcoxon- and t-test, determination of multiple significance levels by Bonferroni-Holmmethod, Fisher´s linear discrimination, leave-one-out and sample splitting and class formation. In the present case a group of patients showing Bronchial Carcinoma and a control group are separated with an error of 0.013. The whole procedure and results of relevant case studies are presented and discussed in detail. The general intention is to enable the investigation of metabolites in human breath as method for early recognition of selected diseases.

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Poster P5 Detection and quantification of microbial metabolites S. Bessenyei, D. Zimmermann, M. Hartmann, A. Grundmann, V. Ruzsanyi, W. Vautz, J. Nolte, J.I. Baumbach ISAS – Institut for Analytical Sciences, Bunsen-Kirchhoff-Str. 11, 44139 Dortmund . Usually, a fungal growth indicates a higher concentration of microbial organic compounds (mVOC). Potential contaminations by different micro-organisms could be indicated by continuous monitoring using ion mobility spectrometers (IMS) and risk assessment procedures will be supported by IMS databases. Ion mobility spectrometry permits a simple and rapid detection of volatile organic metabolites. Therefore, in the present case studies ion mobility spectra were obtained for twenty mVOCs using ion mobility spectrometers equipped with different ionization sources. The detection limits were determined in the range of ng/L using beta-radiation (63Ni sources) and in the low µg/L-range using 10.6 eV discharge lamps as ionization sources for the spectrometers developed. To analyse mixtures, the UV-IMS were combined with gas chromatography and also with multi-capillary-columns (MCC). The advantages and disadvantages of the instrumentation and the practicability of the spectrometer will be discussed in detail for case studies of different volatile metabolites in air. Especially, the metabolites of mold cultures detected using different IMS indicates the practical applicability of the procedure in real cases. New challenges occur not only with respect to the instrumentation, like miniaturisation, but also for rapid classification of analytes. Fast identification and quantification of unknown analytes requires also new calibration procedures as well as new concepts in the field of operation of the spectrometer. Peak-height-diagrams and more dimensional data presentations will be completed by fast classification procedures like cluster analysis and multidimensional calibration methods. The advantages of the method with respect to the operation of the instrument, spectra classification, identification and quantification of gaseous metabolites including examples of relevant and successful applications will be discussed in detail. In addition, robust calibration, identification and quantification procedures especially for field trials of micro ion mobility spectrometers will be considered.

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Poster P6 Tracer studies on human brain proteins by using MALDI-FTICR-MS and LA-ICP-MS after 2D gel electrophoresis J. Susanne Becker1, Miroslav V. Zoriy2, Carola Pickhardt2, Michael Przybylski1, J. Sabine Becker2 1

Central Division of Analytical Chemistry, Research Centre Juelich, 52425 Juelich, Germany. 2Laboratory of Analytical Chemistry, Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany. A new combination of atomic and molecular mass spectrometric methods was developed for the characterization and identification of several metal containing proteins [1]. Human brain protein mixtures were separated by two dimensional (2D) gel electrophoresis and the protein spots were fast screened by microlocal analysis using LA-ICP-MS (laser ablation inductively coupled plasma mass spectrometry) in respect to several elements, such as P, S, Cu, Zn and Fe. Selected protein spots in 2D gel containing these elements were identified after tryptic digestion by matrix assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Results of structure analysis of proteins by MALDI-FTICR-MS were combined with those of the direct element determination in protein spots with LA-ICP-MS in order to characterize proteins in respect to their structure, sequence, phosphorylation state and metal content as well. In order to study the formation of proteins containing Cu, Zn and Fe in a human brain sample isotopic-enriched tracers (54Fe, 65Cu and 67Zn) were doped to two-dimensional gels of separated Alzheimer-diseased brain proteins after 2D gel electrophoresis. The protein spots were screened systematically by LA-ICP-MS, 54Fe/56Fe, 65Cu/63Cu and 67Zn/64Zn isotope ratios in metal-containing proteins were measured directly by LA-ICP-MS. The identification of singular protein spots from Alzheimer-diseased brain separated by twodimensional (2D) gel electrophoresis [2] was attempted by high-resolution biopolymer mass spectrometry (MALDI-FTICR-MS) after excision from the 2D gel and tryptic digestion. References [1] [2]

J. S. Becker, M. Zoriy, U. Krause-Buchholz, J. S. Becker, C. Pickhardt, M. Przybylski, W. Pompe, G. Rödel, J. Anal. At. Spectr. 19 (2004) 1236. J. S. Becker, M. Zoriy, J. S. Becker, C. Pickhardt, M. Przybylski, J. Anal. At. Spectr. 19 (2004) 149.

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Poster P7 Structural determination of neuronal Tau protein using Fourier Transform-ICR-MS J. Su. Becker1, A. Dumitrel1, M. Przybylski1 1

Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany

Tau is a microtubule-associated protein (MAP) and is involved in promoting microtubule assembly and stabilizing microtubulus in vivo. This neurofibrillary, microtubule associated protein has recently gained widespread interest as a target in neurofibrillary tangels characteristic of Alzheimer´s disease and other neuro-degeneretive disorders. The pathophysiologcal aggregation of tau in fibrillary tangels of brain tissue has been shown to be associated to structural changes by multi- or hyper-phosphorylation which has therefore become subject of intensive recent studies. Phosphorylation is probably the most biologically important modification of proteins. Phosphorus concentration in particular proteins can provide information about phosphorylation state [1,2]. Human tau protein was digested with trypsin and analysed by MALDI-/ ESI-FTICR-MS. With MALDI-FTICR-MS a total of 15 serine- and threonine-phosphorylations and complete primary peptide structure could be directly identified. Direct identification of 5 and 6 phosphorylation sites in the multiphosphorylated domains (512 – 528) and (414 – 448) were possible. From the multiphosphorylated domain (512 – 528) 5 and 6 phosphorylated sites could be found. With the ESI-FTICR-MS the complete primary structure including a total of 17 serine- and threonine-phosphorylations could be directly identified from tryptic peptides, partially with very low abundances, which clearly demonstrate the high analytical performance of the precise mass determinations by ESI-FTICR-MS. Mass determination of tryptic peptides provided the direct identification of 7 and 5 phosphorylation sites in the multiphosphorylation domains (512 – 538) and (382 – 398) (T386, S388, T395, T396, S397), respectively [3]. References [1] [2] [3]

M. L. Billingsley, R. L. Kincaid, Biochem. J. 1997, 323 (PT3), 577-591. K. C. Wilhelmsen, Proc. Natl. Acad. Sci. USA 1999, 96, 7120-7121. J.S. Rossier, N. Youhnovski, N. Lion, E. Damoc, J.Su. Becker, F. Reymond, H. Girault, M. Przybylski, Angew. Chem. 2003, 115, 56-60.

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Poster P8 Imaging of metals in human and rat brain tumors by laser ablation induction plasma coupled mass spectrometry C. Pickhardt12, Markus Dehnhardt2 M. V. Zoriy1, Andreas Bauer2, 1 3 2 Andreas Matusch , Guido Reifenberger , Karl Zilles , J. Sabine Becker 1)

Central Division for Chemistry, Research Center Juelich, Germany Institute of Medicine, Research Center Juelich, 52425 Juelich, Germany 3) Institute for Neuropathology, at the Heinrich Heine-University, Duesseldorf, Germany 2)

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to produce images of element distribution in 20 µm thin sections of human and rat brain tissue. The sample surface was scanned (raster area 20 mm x 4 mm) with a focused laser beam (wavelength - 213 nm; diameter of laser crater – 50 µm and laser power density - 3 . 109 W cm-2) in a cooled laser ablation chamber developed for these measurements. The laser ablation system was coupled to a double focusing sector field ICP-MS (Element, Thermo Electron). Concentrations of copper, calcium, zinc and other elements determined in implanted rat brain tumors and human brain glioblastoma multiforme by LA-ICP-MS in primary obtained tumor slice sections in situ. The quantitative determination of element distribution in thin slices of brain tissues was performed using matrix-matched laboratory standards [1]. Whole slices of different tumor brain groups were assessed for ion-species and their concentration and compared to the ion concentration on the contralateral slice side. These results were compared to autoradiographs of adjacent slices with receptorautoradiography for adenosine A1 receptor (A1AR) or PK 11195 (peripheral benzodiazepine-receptor-ligand) and cresyl-violet staining. In preliminary studies, we found a very distinctive distribution of ions, whose levels were decreased within tumors compared to area around tumors and to contralateral side and along the differentially characterized tumor areas: tumor, reactive zone and invasive zone. We conclude from these results, that ion distribution might play a pivotal role in dysregulation of formerly physiological processes, signal transduction and protein function during tumor growth, as has been shown previously for calcium and zinc. References [1] J. S. Becker, M. Zoriy, C. Pickhardt, N. Palomero-Gallagher, K. Zilles, Anal. Chem. 2005 (in press) .

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Poster P9 Capillary electrophoresis ICP-MS for separation of lanthanides in water samples from Israel

C. Pickhardt1, A.K. Malik2, U. Seeling1, A. Izmer1, M. Zoriy1 T. Platzner3, I. Segal3, N. Teplyakov 3, L. Halicz3 and J.S.Becker1 1

Central Division of Analytical Chemistry, Research Center Juelich, D-52425 Jülich, Germany 2 Department of Chemistry, Punjabi University, Patiala-147 002, Punjab, India 3 Geological Survey of Israel, 30 Malkhe Ysrael St., Jerusalem 95501, Israel

An analytical procedure for the determination of lanthanides (Dy, Eu, Er, Yb, Sm and Nd) using capillary electrophoresis isotope dilution inductively coupled plasma mass spectrometer (CE-ID-ICP-MS) in water samples was developed. The water sample was injected into the capillary and was analysed using a double focusing sector field ICP-MS after their separation using Waters electrolyte containing 8-hydroxybutyric acid and creatine acetate at pH 4.2. All the lanthanides are well separated and this permits their analysis without any isobaric interferences. The analytical method developed was applied on several synthetic and natural water samples.

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Poster P10 Determination of Ra-226 in mineral waters at ultratrace level by ICP-MS M.V. Zoriy1,2, Z. Varga3, P. Ostapczuk2, C. Pickhardt1, R. Hille2, J.S.Becker1 1

Central Division of Analytical Chemistry, Research Centre Juelich,D-52425 Juelich, Germany. 2Department for Safety and Radiation Protection, Research Centre Juelich, D52425 Juelich, Germany. 3Institute of Isotopes & Surface Chemistry, Chemical Research Centre of the Hungarian Academy of Science, H-1525, Budapest, Hungary

Determination of long-lived at ultratrace level in analytical chemistry is a challenging task [1, 2]. Among the artificially produced actinides (e.g. 236U, 239Pu, 240Pu, 237Np, 241Am etc) naturally occurred long lived radionuclides such as 226Ra in drinking water and foods is in particular interest [3, 4]. An analytical procedure for determination of 226Ra at low femtogram per ml concentration level in mineral water samples using double focusing sector field ICP-MS (ICP-SFMS) has been proposed. For the pre-concentration and separation of radium from the water sample matrix a tandem of laboratory prepared filter, based on MnO2, and Eichrom “Sr-specific” resin was used. The recovery of the method was found to be 71.5%. Determined limit of detection and limit of quantification for 226Ra were 0.02 and 0.06 fg ml-1 respectively, assuming pre-concentration factor of 10. In addition uranium concentration and uranium isotope ratios were measured. Generally, the radium and uranium concentrations in analyzed mineral water samples were not considerable, except of two samples where the concentration of 226Ra were 10.3 fg ml-1 and 14.2 fg ml-1 and the concentration of U were 17.3 ng ml-1 and 19.2 ng ml-1, respectively. The effective dose of contribution was calculated using the radionuclide concentration and dose conversion factors from the World Health Organization WHO (1993). Assuming the mineral water consumption of 2 l d-1 a slightly higher calculated doses than suggested limit for drinking water (0.1 mSv y-1) in some samples were found.

References [1] [2] [3] [4]

J.S. Becker, H.-J. Dietze, Mass Spectrometry of Long-Lived Radionuclides. In Encyclopedia of Analytical Chemistry, John Wiley. Chichester. 2000. J.S. Becker, Spectrochimica Acta B, 58 (2003) 1757-1784. J.I. Rangel, H. Lopez del Rio, F.M. Garcia, L.L. Torres, M.L. Villalba, L.C. Sujo, M.E.M. Cabrera, Application Radioactiv Isotopes, 56(6) (2002) 931-936. V.N. Epov, D. Lariviere, R.D. Evans, C. Li, R.J. Cornett, J. Radioanal. Nucl. Chem., 256(1) (2003) 53-60.

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Poster P11 Isotope ratio measurements of uranium on biological surface by LA-ICPMS with a cooled laser ablation chamber in microscale range Myroslav V. Zoriy1, M. Kayser2, A. Izmer1, C. Pickhardt1 and J.S.Becker1 1

Central Division of Analytical Chemistry, Research Centre Juelich, D-52425 Juelich, Germany 2 Bioptic laser systems, Berlin, Germany

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a powerful analytical technique for the determination of element concentrations at the trace and ultratrace level and isotope ratio measurements of long-lived radionuclides in solid samples [1, 2]. In addition, this technique can also be applied as the surface analytical technique, in particular, for the surface analysis of biological samples (e.g brain, liver). However, due to the presence of water in different biological sample (more than 30%) the stability of laser ablation process will be altered, that, therefore, leads to decrease of RSD of the measurements [3]. The aim of the present work was to study a method utilizing LA-ICP-MS with cooled laser ablation cell in order to improve precision and accuracy of direct microlocal uranium isotope ratio measurement at the ultratrace level on the surface of biological samples. A cooled laser ablation chamber using a Peltier element was developed in order to analyze element distribution in thin cross sections of frozen tissues with a lateral resolution in the µm range. In order to study the figures of merit of LA-ICP-MS with the cooled laser ablation chamber, one drop (20 µl, U concentration 200 ng mL-1) each of the certified isotope reference materials NIST U350 and U930, the uranium isotopic standard CCLU 500 and also a drop of uranium with a natural isotopic pattern was deposited and analyzed on the biological surface (flower leaf). The precision and accuracy of isotope ratio measurements are significantly improved using cooled laser ablation chamber in comparison to non-cooled chamber. The precision of the measurements of isotope ratios in the range of 2.0-1.6 % for 234 238 U/ U, 1.3 to 0.4 % for 235U/238U and 2.1 – 1.0 % for 236U/238U in selected uranium isotopic standards reference material were determined by microlocal analysis (diameter of laser ablation crater: 15, 25 and 50 µm) using LA-ICP-MS with a cooled ablation chamber. The accuracy of 234U/238U, 235U/238U and 236U/238U isotope ratio measurements varied in the range of 4.2 – 1.1%, 2.4 – 0.5% and 4.8 – 1.1%, respectively, and were dependent on the diameter of the laser beam used. [1] [2] [3]

J.S. Becker, J. Anal. At. Spectrom., 17 (2002) 1172-1185. J.S. Becker, H.-J. Dietze, Mass Spectrometry of Long-Lived Radionuclides. In Encyclopedia of Analytical Chemistry, John Wiley. Chichester. 2000. J. Feldmann, A. Kindness, P. Ek, J. Anal. At. Spectrom., 17 (2002) 813-818.

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Poster P12 Identification of novel proteins in DNA damage signalling pathways using 2D-DIGE I. Bellahn1, M. Szadkowski2, P. Masterson2, J. Prime2, A. Moumen3, S. Jackson3, M. O’Connor2 and D. Carling1 1

MRC Clinical Sciences Centre, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK; 2KuDOS Pharmaceuticals Ltd., 327 Cambridge Science Park, Milton Road, Cambridge CB4 OWG, UK; 3University of Cambridge, Department of Zoology, Downing Street, Cambridge CB2 3EJ, UK

DNA damage is a common event in the life of a cell and may lead to mutation, cancer or cellular death. Amongst DNA damage, double strand breaks (DSB) are one of the most serious problems that the DNA maintenance machinery has to cope with. The DNAdependent protein kinase (DNA-PK) and the Ataxia Telangiectasia Mutated (ATM) kinase, both members of the phosphatidylinositol 3 (PI 3)-kinase-like family, play key roles in the early signalling of DNA damage. Elucidating the signalling mechanism of these pathways will allow us to establish biomarkers of activated DNA repair pathways as well as providing the possibility to identify new drug targets for the treatment of cancer. We have used the powerful method of two dimensional differential gel electrophoresis (2D DIGE) to analyze and quantify changes in protein expression after treatment of cells with ionising radiation (IR), with and without specific inhibitors of the signalling pathways of interest. We have used an experimental strategy that uses a series of internal controls, allowing us to minimize the effects of gel to gel variation and to maximize the accuracy of spot statistics. MALDI-TOF-MS and LC-MS/MS together with MASCOT search engine were used to identify candidate proteins which showed specific changes in their expression profiles. Using genetic and biochemical methods the physiological relevance of these newly identified proteins in the DNA damage response are currently being evaluated. Literatur [1] A. Sancar et al. 2004. Annu. Rev. Biochem. 73:39-85 [2] D. Durocher and S. Jackson 2001. Current Opinion in Cell Biology 13:225-231

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Poster P13 Low level detection of styrene oxide adducts to nucleotides using HPLC/ESI-MS Ulf Bergmann and Michael Linscheid Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin DNA adducts are considered to play a key role in chemical carcinogenesis. An extremely low amount of such DNA damage may result in the formation of cancer. Styrene oxide, which is the main metabolite of the plastics monomer styrene, is known to form DNA adducts and it has proved to generate cancer in rodents [1]. Thus, DNA adducts are an important biomarker and yet the sensitive detection at physiologically relevant levels remains a difficult task. In our ongoing search for methods powerful enough we have made several steps forward to reach this goal; here we present the next combination of techniques to reach relevant detection levels. The large excess of unmodified nucleotides in a digested biological DNA sample compared to alkylated species rules out the direct use of liquid chromatography for the separation without any sample preparation. Employing HPLC/ESI-MS with online preconcentration it was possible to detect 14 styrene oxide adducts among 108 unmodified nucleobases. The calibration was based on phosphorus detection using LC/ICPMS [2, 3]. We now chose to perform an offline solid phase extraction step to remove the large surplus of unmodified nucleotides prior to analysis of the alkylated nucleotides using HPLC/ESIMS. The results obtained are presented on this contribution. Literatur [1] IARC Monographs Vol. 60, Styrene-7,8-oxide, 1994, p. 321 [2] C. Siethoff, I. Feldmann, N. Jakubowski, M. Linscheid; J. Mass Spectrom. 1999, 421 [3] M. Edler, N. Jakubowski, M. Linscheid, Anal. Bioanal. Chem. 2005, in press

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Poster P14 On-line reverse polarity capillary electrophoresis coupled to electrospray quadrupole time-of-flight mass spectrometry for analysis of glycopeptides from Schindler’s disease Laura Bindila, Jasna Peter-Katalinić, Alina Zamfir Institute for Medichal Physics and Biophysics, Universtity of Münster, Germany The hyphenation of the mass spectrometry (MS), in particular electrospray ionization (ESI) MS with high performance separation techniques like capillary electrophoresis (CE)[1-3] is an efficient analytical tool, requiring further development, to analyze highly complex and heterogeneous mixture of carbohydrates. In a previous study a method based on the separation of glycopeptides by CE in the reverse polarity and their detection in the negative ion mode by fully-automated chip based ESI/MS was developed, allowing for increased sensitivity of detection and tandem MS analysis of glycopeptide species [4]. In this contribution, a sheathless on-line CE/ESI MS set-up was optimized to operate in reverse polarity and negative ion mode for MS detection without pressure to assist the sample migration (RPCE/(-)ESI-QTOF MS), coating of the capillaries and/or sample derivatization. Low pH regime employing formic acid at pH 2.8 as a CE buffer system and careful consideration of the separation/detection parameters a constant and stable spray could be obtained by RPCE/(-)ESI-QTOF MS under non-pressure assisted mode. The approach was optimized on a standard mixture of monosaccharides and further applied for screening of a complex glycopeptide mixture originating from the urine of a patient diagnosed with N-acetylhexosaminidase deficiency, known as Schindler’s disease, analyzed before by other strategies. A comparative study upon pressure assisted and non-pressure assisted RPCE/(-)ESI-QTOF MS on this glycopeptide mixture provided clear evidence for a superior separation efficiency and resolution of the RPCE/(-)ESI-QTOF MS over the latter one. A higher number of components (17) could be efficiently resolved under the conditions used, according to the sialylation degree and amino acid/peptide portion. Moreover, due to the high sensitivity and separation power of the novel RPCE/(-)ESI-QTOF MS, previously not detected components, could be identified and their sulfation and O-acetylation modification documented. The potential of this methodology in terms of high sensitivity, separation efficiency, resolution and reproducibility is demonstrated in addition to a new, significantly improved, insight into the sample heterogeneity. References [1] Zamfir, A., Peter-Katalinić, J., Electrophoresis, 2001, 22, 2448-2457. [2] Zamfir, A., Peter-Katalinić, J., Electrophoresis, 2004, 25, 1949-1963. [3] Gennaro, L.A., Harvey, D.J., Vouros, P., Rapid Commun Mass. Spectrom. 2002, 16, 192-200. [4] Bindila, L., Almeida, R., Sterlin, A., Allen, M., Peter-Katalinić, J., Zamfir, A., J. Mass Spectrom. 2004, 39, 1190-1201.

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Poster P15 Towards Understanding the Fragmentation Mechanisms of Protonated Peptides: Competing Pathways for Val-Asn, Val-Gln and Val-Lys Christian Bleiholder 1, Béla Paizs1, Sándor Suhai1 1

Department of Molecular Biophysics, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg.

Mass spectrometry (MS) is a valuable tool for protein identification. Currently, peptide sequencing MS-data analysis tools are based on comparisons of the experimental spectra with in silico predictions for candidate peptides derived from protein and/or nucleic acid databases. Majority of the algorithms only consider the m/z values of the parent and fragment ions neglecting the ion intensity dimension of the spectra. This oversimplification represents a serious drawback in terms of reliability of the whole technique. The reason for this oversimplified treatment, is that the main mass spectrometric event fragmentation of the peptides in the mass spectrometer – is only poorly understood. For more reliable protein identification, semi-quantitative estimates of the relative ion abundances are indispensable. Towards this end the ‘pathways in competition’ (PIC) model [1, 2] has been proposed. This model offers a general framework to cover the major fragmentation pathways of protonated peptides. Moreover, for instance, this model is also capable of explaining the linear free energy relationships (LFER) often reported for ions derived from backbone cleavages [3]. In theory, the flexibility of PIC allows to include all the important peptide fragmentation pathways (PFP) into the model. In this presentation, we discuss some side chain related PFPs and their interaction with PFPs leading to sequence-informative fragment ions. Basic side chains regularly result in the loss of small neutral fragments (H2O, NH3) or the formation of ions, produced by side chain specific reactions [3]. It is evident that these reactions are in competition to the backbone cleavages and, therefore, can potentially disturb LFERs of an, bn, and ym ions. To investigate this issue, we have undertaken a computational study of the model dipeptides Val-Asn, Val-Gln and Val-Lys [4], which have shown to yield mass spectra highly differing from those that can be easily explained based on LFERs [5]. We present here PFPs leading to the loss of water, ammonia and lysine-specific ions. Additionally, we investigate the competition between these side chain specific reactions and the a1/y1- peptide fragmentation pathway [6], which is the major backbone cleavage available to dipeptides. Literature [1] B. Paizs and S. Suhai, Mass Spectrom. Rev., in press, DOI: 10.1002/mas.20024. [2] B. Paizs and S. Suhai, J. Am. Soc. Mass Spectrom., 2004, 15, 103. [3] A. G. Harrison, I. G. Csizmadia, T.-H. Tang and Y.-P. Tu, J. Mass Spectrom., 2000, 35, 683. [4] C. Bleiholder, B. Paizs and S. Suhai, J. Am. Soc. Mass Spectrom., submitted [5] B. Paizs, M. Schnölzer, U. Warnken, S. Suhai and A. G. Harrison, Phys. Chem. Chem. Phys., 2004, 6, 2691. [6] B. Paizs and S. Suhai, Rap. Comm. Mass Spectrom., 2001, 15, 651.

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Poster P16 Präparationsmethoden für hochauflösendes SMALDI Imaging W. Bouschen, O. Schulz und B. Spengler Institut für Anorganische und Analytische Chemie, Justus-Liebig-Universität Gießen Schubertstraße 60, Haus 16, 35392 Gießen SMALDI (Scanning Microprobe Matrix-assisted Laser Desorption Ionisation) Imaging ist eine Methode zur Untersuchung der lateralen Verteilung von biomolekularen Analyten auf natürlichen (z.B. Zellen oder Gewebe) oder synthetischen (z.B. mit immobilisierten Proteinen) strukturierten Oberflächen mit einer Auflösung von etwa 1 µm. Die Signalintensitäten der einzelnen Substanzen werden dabei aus dem Massenspektrum extrahiert und als Graustufenwert innerhalb eines Bildes interpretiert. Entscheidend für die laterale analytische Auflösung ist einerseits der Laserfokusdurchmesser und andererseits die Probenpräparation. Mit dem von uns entwickelten System kann der Laserfokus auf ca. 0.7 µm (gegenüber minimal 30 µm bei kommerziellen Geräten) reduziert werden [1]. Durch Ausnutzung von Flugzeitverschiebungen im ToF-System kann zusätzlich von der Oberfläche eine dreidimensionale Struktur ermittelt werden [2]. Bei räumlichen Auflösungen dieser Größenordnung ist die Probenpräparation der entscheidende limitierende Schritt. Um Migrationseffekte während der Präparation zu minimieren, aber dennoch ausreichende Analyt-Integration in die Matrix zu erhalten wurden verschiedene Methoden, wie z. B. Luftzerstäuben der Matrix oder Bedampfen mit anschließender Rekristallisation [3], untersucht. Eine Aufteilung der Präparation in einen Bedampfungsschritt und einen Umkristallisationsschritt in einer feuchten Atmosphäre erlaubt eine wesentlich bessere Kontrolle über Migration und Integration. Strukturierte biologische und synthetische Oberflächen wurden erfolgreich präpariert, SMALDI-MS untersucht und mit einer neuen automatischen Datenverarbeitung ausgewertet. Diese Untersuchungen zeigen die Einsetzbarkeit und Nützlichkeit von SMALDI-MS im Mikrometerbereich. Das Projekt wurde gefördert vom Bundesministerium für Bildung und Forschung, Aktenzeichen 0312834A. Literatur [1] B. Spengler, M. Hubert; J. Am. Soc. Mass Spectrom. 13 (2002) 735-748. [2] Patentanmeldung, B. Spengler, K.-P. Hinz, W. Bouschen, Aktenzeichen: DE 103 10 518.23 [3] Patentanmeldung, B. Spengler, W. Bouschen, Aktenzeichen: DE 10 2004 019 043.7

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Poster P17 Analysis of cellular protein translocation events using a combined approach of subcellular fractionation, 2D-Polyacrylamide Gel Electrophoresis and MALDI-TOF-MS Jadranka Milosevic1, Patrick Bulau1, Klaus T. Preissner2, Günter Lochnit2, Werner Seeger1 and Oliver Eickelberg1 1

Department of Medicine II, University of Giessen, Aulweg 123, D-35392 Giessen, Germany 2 Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany Hormones of the transforming growth factor  (TGF-) superfamily are involved in the control of development and homeostasis of most tissues in metazoan organisms. TGF- signalling involves ligand-dependent activation of receptor serine/threonine kinases at the cell surface, followed by activation of pathway-specific transcription factors (Smads), nuclear translocation of Smads, and finally modulation of gene expression in the nucleus. Alterations in TGB- activities have been linked to numerous disease states including arteriosclerosis, various forms of human cancer and fibrosis of the kidney, liver and lung. In this contribution we present a proteomic strategy for the characterisation of proteins that undergo cytosolic-nuclear translocation in response to TGB- treatment in lung epithelial cells. After cells were stimulated with TGB- in a time-dependent manner, subcellular fractionation was performed, and cytosolic and nuclear proteins separated by 2DPolyacrylamide Gel Electrophoresis (2D-PAGE). Protein spots of different abundance were subjected to tryptic in-gel digestion and analysed by MALDI-TOF MS. The nuclear enrichment of calgranulin B, creatine kinase, ribonuclearprotein L, and far upstream element binding protein (FUSE), all not detected by immunochemical methods before, was revealed after TGB- treatment. The approach here presented of combined subcellular fractionation, 2D-PAGE and MALDI-TOF MS is a powerful tool to discover new proteins translocation events in biological systems, due to its high resolving power and sensitivity.

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Poster P18 Anwendung von Kryodetektoren in der Flugzeit-Massenspektrometrie P. Christ, F. Pröbst, S. Rutzinger, W. Seidel und S. Uchaikin Max-Planck-Institut für Physik (Werner-Heisenberg-Institut) Föhringer Ring 6, 80805 München Kryodetektoren messen die im Detektor deponierte Energie kalorimetrisch, d.h. im Gegensatz zu MCPs (Multi Channel Plates) unabhängig von der Ionisationswirkung der Moleküle. Dadurch weisen sie jedes auftreffende Molekül unabhängig von dessen Masse bzw. Geschwindigkeit nach. Von ihrem Einsatz verspricht man sich daher eine höhere massenunabhängige Nachweisempfindlichkeit und eine deutliche Erweiterung des Messbereichs von Flugzeit-Massenspektrometern zu höheren Massen hin. Der hier vorgestellte Aufbau besteht aus einem Refletron-TOF mit einer MALDI Ionenquelle kombiniert mit einem Kryostaten der es ermöglicht den Kryodetektor (ein Phasenübergangsthermometer mit einer Fläche von 3x3mm²) bei Temperaturen von 70 - 130mK zu betreiben. Es wird der Status dieses Projektes vorgestellt. An Hand von Verdünnungsreihen konnte gezeigt werden, daß durch den Einsatz von Kryodetektoren die zum Nachweis erforderliche Stoffmenge von Insulin bis unter 1amol veringert werden kann. Dabei wurde eine standard 'dried droplet' Präparation verwendet. Dieses Projekt wurde durch das BMBF-Leitprojekt 'Proteomanalyse des Menschen' finanziert.

125

Poster P19 Mass Spectrometric Characterization of Protein Derivatives Modified with Amino and Thiol Selective Fluorescence Dyes for Use in Proteome Research H. Christoph1, R. Woisch 2, R. Gohlke2, M. O. Glocker1, H.-J. Thiesen1 1) Proteome Center Rostock, University of Rostock, Institute of Immunology, Joachim-Jungius-Str. 9, 18059 Rostock, Germany. 2) GSG Mess- und Analysengeräte GmbH, Bruchsal, Germany. For a comparison of two different protein samples, like synovial fluid and plasma from a patient suffering from rheumatoid arthritis [1], the DiGE method (Difference Gel Electrophoresis) [2] seems of particular advantage. Several dyes have already been used for this purpose, e.g. the cyanine dyes Cy2, Cy3 and Cy5 (A, from Comp. Amersham Biosciences, Little Chalfont, UK) [2]. Nevertheless, the still remaining problem is applying dyes with two different, widely shifted absorption maxima (abs), that at the same time possess quite similar emission intensities and nearly the same possibly low molecular mass. Furthermore, charge distribution after protein modification should not disturb the 2D electrophoresis runnig properties. Also it is of utmost importance that these dyes should label the protein selectively at specific side chain groups. We are presenting our results in comparison to rhodamine maleimide dyes (B, K.-H. Drexhage, University of Siegen and ATTO-Tec Comp., Siegen, FRG) and hemicyanine maleimides as well as their NHS-esters (C, DYOMICS Comp., Jena, FRG) using 1D gel electrophoresis (sensitivity tests). Me Me

tBu

O

Me Me

O

N

+

N Me

N

O N

CO2Et

O

O N

O

+

(CH2)5

N + N

O O

O

O

S N

(CH2)3

O

O

Me Me

O

N

O O

+ ClO4-

A: Cy 5 NHS minimal

B: ATTO-590 maleimide

C: DY-635 NHS-ester minimal The chemical modification procedures using these dyes were first established using model

peptides and proteins and were checked by mass spectrometry. In our studies rhodamines (B) and hemicyanines (C) were found with a protein detection limit of approximately 0.5 ng using recombinant human Macrophage Colony Stimulating Factor  (rhM-CSF ) [3]. The parallel mass spectrometric identification of low quantities of fluorescence-stained proteins has been successful using the Macromizer time-of-flight mass specrometer equipped with a cryodetector. Hence, newest mass spectrometric detector equipment is ready for successfully analyzing intact chemically modified proteins with very low detection limits. [1] A. Sinz, M. Bantscheff, S. Mikkat, B. Ringel, S. Drynda, J. Kekow, H.-J. Thiesen, M. O. Glocker, Electrophoresis 2002, 23, 3445-3456. [2] H. Christoph, K. S. Lilley, M. O. Glocker, H.-J. Thiesen, DiGE-Evaluation with Amino and Thiol Selective Fluorescence Dyes for 2D Gel Electrophoresis of Rheumatoid Arthritis Samples (poster 32), 3rd International BioCon Valley® Congress Fair, Rostock, 2002. [3] M. O. Glocker, B. Arbogast, R. Milley, C. Cowgill, M. L. Deinzer, Proc. Natl. Acad. Sci. USA 1994, 91, pp. 5868-5872.

126

Poster P20 DETEKTION VON 6 SUDAN-FARBSTOFFEN, DIMETHYLGELB UND PARAROT IN GEWÜRZEN UND SOSSEN MIT HPLC/MS/MS Lutz Hartig1, Katrin Hoenicke1, Kristin von Czapiewski2 Eurofins | Wiertz-Eggert-Jörissen, Handelslaboratorium, Stenzelring 14 b, 21107 Hamburg, Germany; 2Applied Biosystems, Frankfurter Str. 129 B; 64293 Darmstadt, Germany, [email protected] Sudane sind rote Farbstoffe, die vor allem zum Färben von Mineralölprodukten und Wachserzeugnissen verwendet werden. Im Sommer 2003 wurde Sudan 1 in verschiedenen Erzeugnissen, beispielsweise in Chili- oder Paprikapulver und Chili- oder Paprikagewürzmischungen nachgewiesen, obwohl der Farbstoff nach europäischem Recht für die Verwendung in Lebensmittel nicht zugelassen ist. Experimentelle Befunde belegen, dass Sudan 1 erbgutverändernde Eigenschaften aufweist. Da die Substanz in Tierversuchen die Substanz krebsauslösend wirkte, wird davon ausgegangen, dass Sudanfarbstoffe möglicherweise auch für den Menschen ein Krebsrisiko darstellen könnten. Das BfR empfiehlt, die Sudan-Farbstoffe in Überwachungsmaßnahmen miteinzubeziehen (Stellungnahme Nov. 2003). In diesem Paper wird eine LC-MS-MS-Methoden vorgestellt, die zum Nachweis von Sudan I bis IV, B, G sowie Dimethylgelb und Pararot geeignet ist. Die vorgestellte Methode ist für Gewürze und Soßen validiert. Am API 2000 TM werden Nachweisgrenzen von 10ug/kg erreicht. Bisher wurden mehr als 500 Proben untersucht mit positiven Befunden für Sudan I, IV, Pararot und Dimethylgelb.

127

Poster P21 Charakterisierung von Glycoproteinen mittels Massenspektrometrie: CEESI-TOF-MS and LC-ESI-IT-MSn U. Demelbauer1, C. Neusüß1, M. Pelzing1, M. Macht1, K. Schubert2 1

Bruker Daltonik GmbH, Permoser Str. 15, 04318 Leipzig, Germany 2 TFH Wildau, Bahnhofstr. 1, 15745 Wildau

Die meisten Glykoproteine zeigen eine ausgeprägte Verteilung unterschiedlicher Isoformen. Die post translationalen Modifizierungen sind abhängig von der Spezies und der Umgebung in der die Spezies lebt. Darüber hinaus ist der Grad der Glykosilierung und die Struktur der Glykane häufig sehr stark für die biologische Wirksamkeit verantwortlich. Die Glykane von Glykoproteinen wurden bislang meist nach Freisetzen der Zuckerstrukturen analysiert. Dies liefert zwar ein detailliertes Bild der Glykane, man verliert jedoch jegliche Information aus welchen Glykanen sich eine spezielle Isoform zusammensetzt und auch welchem Peptid welche Zuckerstruktur zugeordnet werden kann. Enzymatische Spaltung der Proteine zu Peptiden und Glykopeptiden mit anschließender MS (MSn) Analyse liefert die Information über Bindungsstelle im Protein und die zugehörige Glykanstruktur, nicht aber eine umfassende qualitative und quantitative Aussage über eine spezielle Isoform. Dies kann man nur am intakten Protein untersuchen. Wir zeigen hier einen kombinierten Ansatz, der sowohl die Glykopeptide mittels LC-ITMSn als auch die Isoformen auf intakter Proteinebene mittels ESI-TOF MS charakterisiert. Nach tryptischer Spaltung der Proteine wurden die entstehenden Peptide mittels ESI-IT-MS nach Trennung mit HPLC analysiert. Für die MS² und MS³ wurde eine Datenabhängige Precursor Selektion gesetzt. Durch typische Fragmente wurden die Glykopeptide im Chromatogramm identifiziert und mittels MS² und MS³ Spektren die Glycanstruktur und die Peptidsequenz eindeutig bestimmt. Die intakten Proteine wurden mittels CZE getrennt und online mit einem ESI-TOF vermessen. Die hervorragende Trennleistung der CZE, im speziellen von Begleitstoffen wie Salzen, aber auch nicht glykosilierten Proteinen sowie die hohe Auflösung moderner ESITOF Geräte erlaubt es z.B. für Fetuin 46 unterschiedliche Isoformen zu detektieren. Durch Darstellung der geeigneten Ionenspuren kann eine quantitative Abschätzung der einzelnen Isoformen gemacht werden. Mit einem derart kombinierten Ansatz kann man daher die Struktur der Glykane als auch deren Bindungsstelle im Protein bestimmen. Weiterhin wird der integrale Zuckergehalt einzelner Isoformen und deren Quantität im Protein analysierbar. Durch Kombination beider Informationen bekommt man ein umfassendes Bild des Glykoproteins. Dies ermöglicht letztlich Rückschlüsse über Wirkung und Funktion der verschiedenen Isoformen zu ziehen.

128

Poster P22 Host-Bacteria-Interactions analyzed by 2D-PAGE and nanoLC-ESIMS/MS W. Engst, C. Alpert, A. Gühler & M. Blaut German Institute of Human Nutrition Potsdam-Rehbrücke, D-14558 Nuthetal, Germany The human digestive tract is a complex ecosystem, where several hundred bacterial species interact with each other, food components and the host. The gut microbiota fulfills a number of functions in host physiology but is also presumed to contribute to the development of diseases, such as inflammatory bowel disease, colon cancer and allergies. While the influence of the bacteria on the host has been repeatedly demonstrated, the host may also have an influence on the bacteria. At present, knowledge about these interactions is very scarce. Host-bacteria interactions have mostly been investigated with regard to the host response or to activities of pathogenic bacteria. In contrast, we aim to identify reactions of non-pathogenic bacteria, that result from their contact with host-cells, as they occur in the gastrointestinal tract by means of the detection of differentially expressed proteins. In a proteomic approach the response of non-pathogenic human E. coli bacteria on gut epithelial cells (rat IEC-6) was investigated using an in vitro co-culture model system and Nanospray ESI-MS/MS for subsequent identification of differentially expressed proteins. Protein spots of interest were selected after 2D PAGE from expression patterns of cells grown in culture media or in the presence of gut cells. Proteins were digested with trypsin and the resultant peptide mixtures were analyzed on a Waters capillary LC system (CapLC) coupled to a Waters QTOF-Ultima API mass spectrometer. The acquisition of MS/MS spectra was performed in an automated data directed switching between the MS and MSMS mode based upon ion intensity and charge state. Protein identification based on sequence information of the tryptic peptides was achieved using ProteinLynxTM Global Server 2.1 software (Waters corporation) for processing of MS/MS data and subsequent databank searching. The option of inclusion or exclusion of peptide masses in data acquisition and to merge results by ProteinLynxTM Global Server, permitted the identification of additional peptides, thereby increasing the sequence coverage of identified proteins. By means of this method we identified by now nearly 40 E. coli proteins between 12 and 95 kD which were up- or down-regulated in the presence of rat IEC-6 cells. Judging from the distribution of the up-regulated and the down-regulated proteins, it appears that the bacterial cells adjusted the expression of overall protein synthesis functions while the expression of some other enzymes of the glycolytic pathway was enhanced.

129

Poster P23 Demands on HPLC in the Post-Genomics Era H.J. Wirth1, P. Dawes1, E. Dawes1, and B. Pfeffer2 1

SGE International Pty Ltd., 7 Argent Pl., Ringwood, Vic 3134, AUSTRALIA 2 SGE GmbH, Dolivostr. 12, 64293 Darmstadt, GERMANY

With the solving of the human genome, researchers now have the information of all the possible protein sequences in a human body. The expression of these proteins however varies greatly with the type of issue and the state of the body. The situation is further complicated by post-translational modifications of these proteins either in cleavage, glycosylation or phosphorylation. In order to analyze the protein expression in a certain tissue at a given time the field of proteomics emerged. The analytical tasks faced in Proteomics turned out to be far more complex than the solving of the genome. The difficulty in analyzing protein expression arises from the dynamic nature of protein expression and the large degree of variation in the abundance of individual proteins. While 2D electrophoresis is a commonly used approach to solve the protein content of a tissue sample, capillary LC coupled with multidimensional MS is becoming increasingly popular, because of the ease of automation and higher sample throughput. The complexity of the typical sample demands a multidimensional approach to the LC separation. The LC system can contain any combination of the following columns: Affinity traps for the depletion of high abundance proteins (e.g. hSA accounts for ~60% of the protein content in serum) or the enrichment of a class of proteins of special interest (e.g. metal chelates for the enrichment of phosphorylated peptides), SCX trap columns for a low resolution preseparation in 2D-LC, C4/C8 desalting columns and C18 analytical columns for a high resolution second dimension separation. If the protein glycosylation is of interest, then the collection of columns is extended by a graphitized carbon column, which shows excellent selectivity for the separation of oligosaccharides. At the same time column dimensions are decreasing to gain the necessary sensitivity, 75µm ID columns are now standard. The associated issues with small volumes, sample size and flow rates, provide a unique challenge for column manufacturers. While traditional analytical chromatography is very forgiving in terms of void volumes in capillary- and nano flow LC any amount of void volume and disturbance in the flow path has a detrimental effect on the column performance and with it on the resolution and sensitivity of the separation. At SGE, we are trying to provide a comprehensive approach to nano LC with special regards to minimizing sample dispersion from void volumes, disturbances in the flow path and non-specific interaction with surfaces within this flow path.

130

Poster P24 Investigation of Noncovalent Base Pairing of Nucleobase Functionalized -Peptides using ESI-FT-ICR-MS H. Frauendorf, N. Diezemann, P. Chakraborty and U. Diederichsen Institute of Organic and Biomolecular Chemistry, Georg-August-Universität Göttingen, Tammannstr. 2, D-37077 Göttingen -Peptides have the ability to form helices. In fact only six -amino acids can be sufficient for specific helix formation with every third amino acid being placed on the top of each other. Substituting one side chain out of three of a -peptide helix with the nucleobases adenine, thymine, guanine, or cytosine gave oligomers that have the ability of dimer formation [1]. Four different oligomers only varying in the sequence of the nucleobases (TGAT, TAGT, ATCA, ACTA) were synthesized [2] and submitted to study noncovalent base pairing using ESI-FT-ICR mass spectrometry in aqueous ammonium acetate buffer. Formation of heterodimers can be observed for antiparallel pairing of -peptides (TAGT/ATCA and TGAT/ACTA) in the electrospray mass spectra confirming specific formation of noncovalent complexes. Parallel pairing of the systems TGAT/ACTA and TAGT/ATCA shows dimer formation with significantly lower intensity. So, in the case of -peptide helices antiparallel double strand formation seems to be preffered in comparison to parallel double strand formation. Furthermore, self-pairing effects leading to homodimer formation were discussed. These results are in good agreement with data obtained from other spectroscopic techniques such as temperature dependent UV spectroscopy and CD spectroscopy [2] to show the potential of electrospray mass spectrometry in studying noncovalent interactions at such complexes.

Figure 1: Noncovalent complex of two nucleobase functionalized -peptides

Literatur [1] A. M. Brückner, P. Chakraborty, S. H. Gellman, U. Diederichsen, Angew. Chem. 2003, 115, 4532 [2] P. Chakraborty, U. Diederichsen, Chemistry, submitted.

131

Poster P25 Anwendung der ESI-FT-ICR-Massenspektrometrie zur Untersuchung des Startvorganges radikalischer Polymerisationen M. Buback1, H. Frauendorf 2, F. Günzler1 und P. Vana1 1 Institut für Physikalische Chemie der Georg-August-Universität Göttingen, Tammannstr. 6, D-37077 Göttingen 2 Institut für Organische und Biomolekulare Chemie der Georg-August-Universität Göttingen, Tammannstr. 2, D-37077 Göttingen Bei radikalischen Polymerisationen sind genaue Kenntnisse der Kinetik, insbesondere des Startschrittes, und der aus den Starterverbindungen entstehenden radikalischen Spezies für die Produkteigenschaften von großer technischer Bedeutung. Dabei können in Abhängigkeit von den als Startermaterialien eingesetzten Substanzen verschiedene Folge-, Übertragungsund Terminierungsreaktionen ablaufen, wodurch eine Vielzahl an unterschiedlichen radikalischen Spezies gebildet wird, welche als Endgruppen in das gebildete Polymer eingebaut werden. Kürzlich konnte gezeigt werden, dass die ElektrosprayMassenspektrometrie eine hervorragende Methode zur Analyse der Kinetik und des Mechanismus von Radikale bildenden Startreaktionen durch die Endgruppenbestimmung von synthetischen Polymeren darstellt [1]. Für die umfassende Charakterisierung dieser komplexen Gemische kommt den durch die hochauflösende ESI-FT-ICRMassenspektrometrie erhaltenen exakten Molekül-massen, die die Ermittlung der Elementarzusammensetzung erlauben, eine herausragende Bedeutung zu. In den vorgestellten Untersuchungen wurden Polymethylmethacrylate, die durch mit verschiedenen organischen Peroxiden gestartete radikalische Polymerisation gewonnen wurden, auf ihre Endgruppen mittels niederauflösender, aber auch hochauflösender ESI-MS detailliert analysiert. Die FT-ICR-MS-Messungen erfolgten durch direkte Infusion der gelösten Produkte in verschiedenen Lösungsmitteln. Mittels interner Kalibrierung gelingt eine sehr weitreichende Produktanalyse in dem interessierenden Molekulargewichtsbereich von m/z 300 bis 1000, wobei den meisten nachgewiesenen Spezies unter Berücksichtigung der Ausgangsverbindungen eine eindeutige Elementarzusammensetzung zugeordnet werden kann. Die Fehler der Massenbestimmung liegen dabei trotz der Komplexität der Gemische und unterschiedlicher Intensitäten im allgemeinen unter 1 ppm. Mit dieser Methode konnten eine Reihe von bisher unbekannten Zwischenprodukten bei der Methylmetacrylatpolymerisation identifiziert und tiefergehende Kenntnisse über die ablaufenden Prozesse erhalten werden.

Literatur [1] M. Buback, H. Frauendorf, P. Vana, Initiation of Free-Radical Polymerization by Peroxypivalates Studied by Electrospray Ionization Mass Spectrometry, Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 42, 4266-4275 (2004)

132

Poster P26 Simultaneous ESI/APCI Acquisition Using a Multimode Source in High Throughput Analysis Friedrich Mandel 1, Patrick D. Perkins 2, Steven M. Fischer 2, Douglas E. McIntyre 2, Wayne P. Duncan 2 1

Agilent Technologies, Waldbronn, Germany,

2

Agilent Technologies, Palo Alto, USA

For high throughput analysis, fast analysis time is critical. A major problem is that the analyte may not respond in one ionization mode (ESI, APCI, APPI) and therefore must be reanalyzed using a different ionization mode. Unfortunately, this usually means exchanging the ion source and reanalyzing the sample. An ideal source would operate in more than one ionization mode simultaneously. It would use ESI as one of its modes and furthermore, such a source would function at the high flow rates typical of high throughput LC/MS analysis. We have developed a single ionization source that operates simultaneously in ESI and APCI modes, or in each mode if desired, and this work describes its performance when analyzing test compounds for high throughput analysis. The multimode source incorporated parts of both ESI and APCI sources along with some novel elements that permitted both ionization modes to be performed independently or simultaneously. The HPLC effluent was nebulized with a single sprayer at ground potential. The droplets emerged into an electric field generated by a charging electrode and produced electrospray ions, and these ions were drawn towards the capillary and into the mass spectrometer. The residual droplets passed through a field-reversing electrode that separated the ESI portion of the source from the APCI portion. The droplets were exposed to radiation from two infrared lamps emitting at the absorption frequency of water, which caused complete vaporization of the effluent and produced the proper gas phase conditions for APCI. The vapor and analytes were then ionized in the presence of a corona needle, and the ions formed were drawn from the bottom of the source towards the capillary. The novel elements and their use (patents applied for) permitted ESI and APCI to proceed simultaneously and optimally in different regions of the same source, without switching voltages or flows, even at high HPLC flow rates and high aqueous mobile phase composition. A test suite of compounds was run in scan mode using positive/negative switching on a dedicated ESI source, a dedicated APCI source, and the multimode source, and the results are demonstrated. Different hardware and operating parameters were examined to determine their effects on sample throughput. Adding overlapped injection and reducing the gradient delay volume improved throughput by about 29%. Adding alternating column regeneration yielded an additional 28% improvement. Finally, a factor of 2× improvement was gained by using the multimode source for simultaneous ESI and APCI instead of dedicated ion sources. The final analysis time to obtain positive and negative ESI and APCI analysis for a 96 well plate was just under three hours.

133

Poster P27 GC/MS analysis of disease markers the in breath of critically ill patients Fuchs Patricia, Miekisch Wolfram, Noeldge-Schomburg Gabriele FE, Schubert Jochen K University of Rostock, Clinic of Anaesthesiology and Intensive Care, Schillingallee 35, 18057 Rostock, Germany; Tel +49-0381-494-5955, Fax +49-0381-494-6412. Background: Volatile organic compounds (VOC´s) are produced anywhere in the body and are transported via the blood stream and exhaled through the lung. They can reflect physiological or pathological biochemical processes, such as lipid peroxidation, liver disease, renal failure, allograft rejection, dextrose or cholesterol metabolism. Due to the very low concentrations of endogenous VOC´s in exhaled air sophisticated technical procedures are necessary for breath analysis. In critically ill patients the problem is further aggravated by numerous contaminants from the clinical environment and from the ventilator. Nevertheless, breath tests are attractive because they are non-invasive and can be repeated frequently without any risk for the patient. This is especially important in the dynamically changing state of critically ill patients. In our studies we used different methods of pre-concentration and GC/MS to detect VOC´s in the breath of mechanically ventilated patients. Methods: 44 (first study (S1)) and 46 (second study (S2)) critically ill and mechanically ventilated patients of an interdisciplinary intensive care unit (ICU) were investigated. Patients suffered from (S1) ARDS (lung injury) and head injury and (S2) pneumonia, sepsis, SIRS (systemic inflammatory response syndrome) and multiple trauma. Breath samples were drawn from the respiratory circuits by means of a gas tight syringe. Alveolar gas was taken near the endotracheal tube (ETT) under control of expired CO2. Inspiratory samples were taken near the ventilator inlet. Volatile substances in breath were pre-concentrated by means of sorbent traps or SPME, thermally desorbed and analyzed by GC. Substances where identified by mass spectrometry (MS) and quantified using the ion fragments m/z 67 (isoprene), 43 (n-pentane), 149 (isoflurane) and 58 (acetone).For the second study 3 mL of arterial and mixed venous blood were taken from each patient at exactly the same time as breath samples were collected, preconcentrated by headspace SPME and analyzed as described above. Results: Lower limits of quantitation (LLOQ) and precision (% RSD) were 0.05 (4.4), 0.10 (3.8), 0.02 (2.5), and 0.08 (5.9) nmol L-1 for the determination of isoprene, n-pentane, isoflurane and acetone in blood. LLOQs for the analysis of isoprene, n-pentane, isoflurane and acetone in exhaled gas were 0.01, 0.02, 0.02, and respectively 0.05 nmol L-1. Intraday variation was < 5% RSD (n = 8) for all substances. S1: Expired isoprene concentrations in ARDS patients were significantly lower (p<0,002) than in patients with head injury. Pentane concentrations showed a tendency to be higher in ARDS patients, acetone concentrations were not different in both patient populations. S2: Patients with sepsis had higher n-pentane and lower acetone concentrations in mixed venous blood than patients without sepsis 2.3 (0.4-8.7) vs. 0.6 (0.3–1.5) nmol L-1 and 69 (22-99) vs. 18 (6.7–56) µmol L-1). N-pentane and acetone concentrations in breath showed no differences between the patient groups, regardless whether or not expired concentrations were corrected for inspired concentrations. Conclusion: Breath analysis is a promising non-invasive tool for diagnosis and surveillance in critically ill patients. SPME technique simplifies and speeds up GC/MS analysis of volatile organic compounds in breath considerably. In mechanically ventilated patients, concentration profiles of volatile substances in breath may considerably deviate from profiles in blood when inspired concentrations are high (>5%).

134

Poster P28 Massenspektrometrische Untersuchung der Michael-Addition von Malonsäurediethylester an Acrylnitril: Direkter Nachweis von transienten Carbanionen Sven Fürmeier 1 und Jürgen O. Metzger 2 1

Institut für Chemie, Universität Kassel, Heinrich-Plett-Str. 40, 34132 Kassel. 2 Institut für Reine und Angewandte Chemie, Universität Oldenburg, Postfach 2503, 26111 Oldenburg.

Die Michael-Addition ist eine präparativ wichtige Reaktion, in der C-H-acide Verbindungen wie Malonester an ,-ungesättigte Carbonylverbindungen wie Acrylester, Methylvinylketon und Acrylnitril unter Knüpfung einer neuen C,C-Bindung addiert werden. Als repräsentatives Lehrbuchbeispiel wurde die Umsetzung von Malonsäurediethylester (1) mit Acrylnitril (3) in Gegenwart einer Base zum Bis(2-cyanethyl)malonsäurediethylester (8)[1] mittels der Mikroreaktor-gekoppelten Elektrospray-Ionisations-Massenspektrometrie[2] untersucht (Schema 1). Das durch Reaktion von 1 mit Natriumethylat gebildete stabile Malonsäurediethylester-Anion (2) addiert an 3 zu dem transienten Carbanion 4. Durch Protonierung von 4 entsteht das Zwischenprodukt 2-Cyanethylmalonsäurediethylester (5); Deprotonierung durch die Base gibt das stabile Carbanion 6. Die zweite Michael-Addition an Acrylnitril über das transiente Carbanion 7 gibt schließlich Produkt 8. Durch die Mikroreaktor gekoppelte ESI-MS konnten nebeneinander Edukt 1, Zwischenprodukt 5, Produkt 8, die stabilen Malonationen 2 und 6 und insbesondere das unter Quasistationaritätsbedingungen in sehr geringer Konzentration vorliegende transiente Carbanion 7 in der reagierenden Lösung massenspektrometrisch beobachtet und charakterisiert werden. N

N O

O O

EtO-

O

- EtOH

O

3

O

N

H+ O

O

O O

1

N

O 2

EtOO - EtOH O

O

O

O

4

O

O

5

N

O

H+

O O

8

O 6

N O

O

N O O

3

O O

N

7

N

Schema 1. Michael-Addition von Malonsäurediethylester (1) an Acrylnitril (3) zum Bis(2-cyanethyl)malonsäurediethylester (8) über das Zwischenprodukt 2-Cyanethylmalonsäurediethylester (5) und die Carbanionen 2, 4, 6 und 7. Literatur [1] Autorenkollektiv, Organikum, 21. Auflage, Wiley-VCH Verlag, Weinheim, 2001, S. 601. [2] S. Fürmeier, J. O. Metzger, J. Am. Chem. Soc. 2004, 126, 14485-14492.

135

Poster P29 Mass spectrometric characterization of peptides resulting from enzymatic digestion of recombinant human tropoelastin Melkamu Getie1, Christian E.H. Schmelzer1, Anthony S. Weiss2, Reinhard H.H. Neubert1 1

Institute of Pharmaceutical Technology and Biopharmaceutics, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck Str. 4, 06120, Halle (Saale), Germany 2 Department of Molecular Biotechnology, University of Sydney, NSW 2006, Australia

Elastin is an important component of elastic fibers that provide elasticity to organs, such as skin, lung, aorta, and ligaments. Pathological disorders, such as solar elastosis, emphysema, aneurysms and atherosclerosis, in which the mechanical and elastic properties of tissues are altered, are associated with abnormalities in elastic fibers [1,2]. Understanding the biochemical basis of such disorders requires information about the primary structure of elastin. However, due to its extreme insolubility, obtaining such information has been difficult. Previous studies were mainly focused on complete hydrolysis of the protein in strong acidic environment and analysis of the resulting cross-linked amino acids, desmosine and its isomer isodesmosine, which are only found in elastin. This approach has been challenged, among other things, by the incomplete separation of the amino acids with the analytical methods employed and the partial information that can be obtained about the entire elastin molecule only based on the cross-linked amino acids. Extraction of elastin from a healthy and diseased tissue and comparison of the molecular structures of the peptides resulting from enzymatic digestion of the protein could give a better image of the biochemical changes that occur due to the mentioned pathological conditions. Therefore, in this work, we determined the sequences of peptides resulting from digestion of an elastin precursor, synthetic recombinant human tropoelastin lacking exon 26A (SHELdelta26A), with pepsin. Tandem mass spectrometry experiments were performed on selected peptide ions from the resulting peptide mixture using LC-ESI and/or nano-ESI mass spectrometry and the sequences of the peptides were determined by database matching and/or by de novo sequencing. Accordingly, about 83% of the sequence on the GenBank entry AAC98394 (amino acid residues 27 to 724), based on which the recombinant tropoelastin was synthesized [3], was covered by the sequence of about 100 peptides identified. Literature [1] L.B. Sandberg, N.T. Soskel, J.C. Leslie, N. Engl. J. Med., 304 (1981) 566-579. [2] T.M. Olson, V.V. Michels, Z. Urban, K. Csiszar, A. Christiano, D.J. Driscoll, R.H. Feldt, C.D. Boyd, S.N. Thibodeau, Hum. Mol. Genet., 4 (1995) 1677-1679. [3] W.J. Wu, B. Vrhovski, A.S. Weiss, J. Biol. Chem., 274 (1999) 21719-21724.

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Poster P30 A novel strategy for identification of protein-DNA contacts by MALDITOF-MS/MS Hildegard Geyer1, Rudolf Geyer1 and Vera Pingoud2* 1

Biochemisches Institut, Justus-Liebig-Universität, Friedrichstraße 24, D35392 Giessen, 2Institut für Biochemie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen

Protein-DNA interactions are involved in many cellular processes. Sequence-specific DNAbinding proteins have been identified and their interactions with DNA have been intensively studied. To understand how these proteins function, it is essential to identify the contacts between proteins and DNA. The classical approach for the identification of the cross-linked peptide or amino acid residue is sequencing by Edman degradation. In parallel, mass spectrometry has been used in cross-linking studies for the determination of the molecular weight and for amino acid sequencing. Its principal advantage is higher sensitivity and specificity but enrichment of the peptide-oligodeoxynucleotide adduct is necessary for the successful analysis by MS. The majority of published protocols dealing with the identification of protein-DNA contacts use liquid chromatography and/or polyacrylamide gel electrophoresis for purification of peptide-oligodeoxynucleotide adducts from peptides and/or free oligodeoxynucleotides prior to mass spectrometry. These protocols, however, suffer from a low sample recovery which implies that large amounts of a purified protein are required. In this study we wanted to improve the sensitivity of the analysis of photocross-linked protein-DNA adducts by mass spectrometry. We describe a novel strategy to localize crosslinked amino acid residues that combines laser-induced photocross-linking of the protein with a 5-IdU-modified oligodeoxynucleotide, proteolytic digestion of the peptideoligodeoxynucleotide heteroconjugate, Fe3+-IMAC (immobilized metal affinity chromatography) purification and hydrolysis by HF with efficient MALDI-TOF mass spectrometry. Proteolytic digestion produces an peptide-oligodeoxynucleotide adduct the apparent molecular mass of which can be easily determined by MALDI-TOF-MS. After its separation from non-cross-linked peptides by Fe3+-IMAC (together with free oligodeoxynucleotide) the oligonucleotide part is hydrolysed by treatment with HF and the remaining nucleoside-modified peptide can be directly analysed by MALDI-TOF-MS and -MS/MS. This method is suitable for the fast identification of the site of contact between proteins and nucleic acids starting from picomole quantities of cross-linked complexes [1,2]. Literatur [1] Geyer, H., Geyer, R., et al. (2004) A novel strategy for the identification of protein-DNA contacts by photocrosslinking and mass spectrometry. Nucl. Acids Res., 32, e132. [2] Pingoud, V., Sudina, A., et al. (2005) Specificity changes in the evolution of type II restriction endonucleases: a biochemical and bioinformatic analysis of restriction enzymes that recognize unrelated sequences. J Biol Chem, 280, in press.

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Poster P31 Fully Automated Liquid Injection Field Desorption/Ionization (LIFDI) Mass Spectrometry for High Throughput Screening of Compounds with Various Polarity Jens Griep-Raming 1, H. Bernhard Linden 2 1

Thermo Electron Corp., Bremen, Germany. 2 Linden CMS, Leeste, Germany.

Although field desorption/field ionization (FD/FI) is a technique that has been in use with mass spectrometry (MS) for a long time already, it found broad applications only in the seventies when FAB, MALDI, and ESI were not yet introduced. Nowadays FD/FI is limited to niche applications, such as hydrocarbon analysis. The fact that FDMS almost exclusively yields M+. ions for virtually any compound class, makes it attractive to analyze compounds that are prone to extensive fragmentation in an unspecific way, and would not give good results with other ionization techniques. However, the extremely difficult handling of the FD emitters, and their fragility made many researchers to refuse using the technique. With the introduction of an in-vacuum liquid samples deposition technique (liquid injection field desorption/ionization, LIFDI)1-2, these limitations were greatly removed, and the application of FD extended to FT-ICR MS including continuous flow FD operation 3. Up to now the technique has only been applied with manual sample introduction. Here we report on the use of a specifically modified automated liquid sampling system coupled to the LIFDI ion source installed on a Finnigan MAT 95 XP mass spectrometer. A setup has been implemented based on a CTC PAL autosampler that allows to automatically puncture autosampler vial septa, then insert a coaxially guided fused silica capillary into the vial to pick up the sample solution, and finally analyze the sample. It could be shown that the automated technique leads to increased throughput compared to manual sample introduction, is able to detect and compensate for carry over, if necessary, and can hence provide unattended analyses of quite different samples. Literature [1] Linden H B and Maurer M, In-source sample infusion for fully automated FD MS, Proceedings of the 48th Annual Conference on Mass Spectrometry and Allied Topics, Long Beach, CA 2000, WPA 050 [2] Linden H B, Liquid Injection Field Desorption Ionization: a new Tool for Soft Ionization of Samples including Air Sensitive Catalysts and Non-Polar Hydrocarbons, Eur. Mass Spectrom. 10 (4), 459-468 (2004) [3] Schaub T M, Linden H B, Hendrickson C L, Marshall A G : Continuous Flow Sample Introduction for Field Desorption/Ionization Mass Spectrometry, Rapid Commun. Mass Spectrom. 18 (14), 1641-1644 (2004)

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Poster P32 REMPI-spektroskopische Untersuchungen an Dichlorbenzolen Angela Gaber1, Zekeriya Altug1 und Jürgen Grotemeyer1 1

Inst. f. Phys. Chem. der CAU zu Kiel, Olshausenstr. 40, 24098 Kiel, [email protected]

Die resonanz verstärkte Multiphotonen-Ionisation (REMPI) - Spektroskopie ist eine sehr leistungsstarke Methode zur Spurenanalytik. Diese zweidimensionale Messtechnik verbindet Messschnelligkeit mit Sensitivität und Selektivität der Analyse. Die REMPISpektroskopie tastet den ersten angeregten Schwingungszustand des zu untersuchenden Moleküls ab. Dieser besitzt ähnlich dem Schwingungsgrundzustand in der IR-Spektroskopie Fingerprint-Qualität, allerdings sind hierbei die Unterschiede zwischen isomeren Verbindungen sehr viel stärker ausgeprägt, so dass diese unterschieden werden können. Die drei Konstitutionsisomere des Dichlorbenzols sind starke Umweltgifte. Sie unterscheiden sich allerdings untereinander in ihrer Gefährlichkeit dem Menschen gegenüber. Eine Unterscheidung bzw. Charakterisierung der drei Verbindungen in einem potentiellen Stoffgemisch ist daher von großem Interesse. Mit Hilfe der REMPISpektroskopie ist eine Identifizierung der Substanzen möglich. Aufwendige Auftrenn- und Reinigungsschritte sind bei diesem Verfahren somit überflüssig und erlauben daher eine schnelle Spurenanalyse. Die durchgeführten Messungen zeigen große Unterschiede in den REMPI-Spektren der drei isomeren Dichlorbenzole. Dabei wurden neben Ein-Farben (1C) - auch Zwei-Farben (2C) Spektren aufgenommen, da der 0-0-Übergang der drei Verbindungen jeweils oberhalb der halben Ionisationsenergie liegt. Die Zuordnung der beobachteten Banden der aufgenommenen Schwingungsspektren erfolgte durch den Vergleich mit den Ergebnissen ebenfalls durchgeführter ab-initio Rechnungen und mit Hilfe von bereits veröffentlichten Ergebnissen [1], [2]. Bei den durchgeführten Rechnungen zeigte sich, dass der S1-Zustand sich am besten mit Hilfe von TD-SCF/6-311++G(d,p) bzw. CASSCF(6,6)/6-311++G(d,p)Rechnungen beschreiben läßt. Literatur: [1] E. Rohlfing, C. Rohlfing, J. Phys. Chem. 1989, 93, 94-101. [2] W. Sands, R. Moore J. Phys. Chem.1989, 93, 101-107.

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Poster P33 Mapping nucleotide induced conformational changes in the Escherichia coli Hsp70 chaperone DnaK using amide hydrogen exchange and mass spectrometry Christian Graf , Wolfgang Rist , Bernd Bukau und Matthias P. Mayer Zentrum für Molekulare Biologie Heidelberg (ZMBH), Universität Heidelberg. Hsp70 chaperones assist many protein folding processes including de novo folding of polypeptides, refolding of stress denatured proteins, and control of stability and activity of transcription factors and protein kinases. Central to the mechanism of Hsp70s are nucleotide-induced changes in binding affinities for their substrates. ATP binding to the ATPase domain increases substrate association and dissociation rates in the substrate binding domain by 2 and 3 orders of magnitude, respectively, leading to a decrease in substrate affinity of 10 to 20-fold. Structural information is only available for the two individual domains in the high affinity state and it remains elusive, which conformational changes lead to the low affinity state. In order to investigate these conformational changes, we performed amide hydrogen exchange experiments combined with mass spectrometry using a new online-HPLC setup. To analyze deuteron incorporation in the E. coli Hsp70 chaperone DnaK, we used the ATPhydrolysis deficient DnaK-T199A mutant in presence or absence of different nucleotides. Deuteron incorporation into full-length DnaK-T199A in the absence and presence of ATP shows that two counteracting processes occur upon ATP binding, part of the structure becomes less solvent accessible while another part becomes more solvent accessible. Time-dependent deuteration on the peptide level was analyzed after digestion of the deuterated protein by immobilized pepsin. It reveals that specific regions in the ATPase domain become significantly less solvent accessible in the presence of ATP while regions in the substrate-binding domain become more flexible. Exchange experiments were also carried out in presence of substrate peptides and proteins to localize conformational changes in DnaK upon substrate binding. Taken together, these data give for the first time an insight into the dynamics of Hsp70 proteins thereby improving our understanding of how these chaperones control the folding status of their substrates.

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Poster P34 Pigmentvergleich der rezenten Seelilie Hypalocrinus naresianus mit 240 Millionen Jahren alten Fossilien von Carnallicrinus carnalli Klaus Wolkenstein 1, Jürgen H. Gross 2, Matthias Pelzing 3 , Uwe Demelbauer 3 und Heinz F. Schöler 1 1

Institut für Umwelt-Geochemie der Universität, Im Neuenheimer Feld 236, D-69120 Heidelberg. 2 Organisch-Chemisches Institut der Universität, Im Neuenheimer Feld 270, D-69120 Heidelberg. 3 Bruker Daltonik GmbH, Permoserstr. 15, D-04318 Leipzig. In der Vergangenheit präsentierten wir basierend auf Arbeiten Blumers[1] Untersuchungen zur Identifizierung von Farbstoffen in fossilen Seelilien aus der Trias (Carnallicrinus carnalli), die zeigten, daß organische Pigmente nahezu unverändert 240 Millionen Jahre überdauern können.[2] Als Farbstoffe wurden das heute im Johanniskraut (Hypericum perforatum) vorkommende, medizinisch bedeutsame Hypericin[3] und damit strukturverwandte Substanzen identifiziert. Für die Biologie ist es nun auch von Interesse, rezente Seelilien mit ihren längst ausgestorbenen Vorfahren zu vergleichen, um beispielsweise chemotaxonomisch Verwandschaftsverhältnisse aufzuklären. Seelilien gehören wie Seeigel und Seesterne zur Gruppe der Stachelhäuter. Ein Exemplar der in ca. 800 m Meerestiefe lebenden rezenten Seelilie Hypalocrinus naresianus wurde uns freundlicherweise von Prof. Tatsuo Oji (Universität Tokyo, Japan) zur Verfügung gestellt. Erste massenspektrometrische Untersuchungen eines Rohextraktes werden hier vorgestellt. Der dunkelgrüne ethanolische Extrakt des ganzen Tieres wurde in orientierenden Versuchen mit MALDI und ESI untersucht. Das negativ-Ionen ESI-Spektrum zeigte zwischen m/z 820 und m/z 1010 elf Ionen, sechs drei- und und fünf vierfachbromiert, deren m/z-Werte teils mit bekannten Farbstoffen aus der Gruppe der Gymnochrome aus Gymnocrinus richeri übereinstimmten.[4] Hochauflösende ESI-TOF-Spektren lieferten die eindeutige Zuordnung aller Summenformeln (C34H20Br3O10 bis C40H29Br4O11) bei sehr guter Übereinstimmung von experimentellen und berechneten Isotopenmustern. ESI-MS/MS lieferte Abspaltungen, die mit den vermuteten Strukturen gut korrelieren. Vergleiche von LDI mit MALDI unter Verwendung verschiedener Matrizes ergab, daß nur Dithranol geeignet ist. Für künftige Arbeiten zur Isolierung und vollständigen Charakterisierung aller Farbstoffe von Hypalocrinus naresianus wollen wir MALDI mit Dithranol-Matrix zum probensparenden Screening von Extrakten und chromatographischen Fraktionen einsetzen. Alle Massenspektren wurden im negativ-Ionen Modus aufgenommen: MALDI-TOFSpektren an einem Bruker Biflex mit Pulsed Ion Extraction, ESI- und ESI-MS/MS an einem Finnigan TSQ700 Triplequadrupolgerät und HR-ESI an einem Bruker micrOTOFfocusGerät. Literatur [1.] Blumer, M. Fossile Kohlenwasserstoffe und Farbstoffe in Kalksteinen. Mikrochemie 1951, 36/37, 1048. [2.] K. Wolkenstein, J. H. Gross, S. Giesa, H. F. Schöler, Nachweis eines Fringelit-Typ Farbstoffs in fossilen Seelilien der Art Chelocrinus carnalli mittels LR- und HR-ESI-MS, ESI-MS/MS und LDITOF-MS. P22, 34. DGMS-Tagung, München, 2001. [3.] Falk, H. Vom Photosensibilisator Hypericin zum Photorezeptor Stentorin – die Chemie der Phenanthroperylenchinone. Angewandte Chemie 1999, 111, 3306. [4.] F. De Riccardis, M. Iorizzi, L. Minale, R. Riccio, B. Richer de Forges, C. Debitus, Journal of Organic Chemistry 1991, 56, 6781.

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Poster P35 Liquid Injection Field Desorption Ionisation am Flugzeit-Massenspektrometer (LIFDI-TOF-MS) Karl-Heinz Gunzelmann, Hans-Martin Hutmacher, Joachim Richert, Rainer Wolf, BASF Aktiengesellschaft, Kompetenzzentrum Analytik, Spektroskopie - GKA/S, 67056 Ludwigshafen Feld-Desorption (FD) ist eine vergleichsweise alte Ionisationstechnik in der Massenspektrometrie. Abgeleitet im Jahr 1969 aus den Vorläufern Feld-Ionen-Mikroskopie (FIM) und Feld-Ionisation (FI) durch H.D. Beckey, repräsentierte FD den Versuch, polare und schwerflüchtige Festkörper möglichst schonend zu ionisieren. Die schwierige Handhabung der Methode in Bezug auf Herstellung, Justage und Haltbarkeit der erforderlichen filigranen FD-Emitter war verantwortlich für ihre geringe Robustheit. Deshalb, aber auch bedingt durch die raschen Erfolge der ESI- und MALDI-Technik, sind FD-Systeme vergleichsweise wenig verbreitet. Die Kombination der FD-Ionenquellen mit Sektorfeldgeräten war lange Zeit die Standard-Instrumentierung. Mittlerweile ist die Methode auch als Option für hochauflösende FT-MS-Geräte erhältlich. Neue Impulse erhielt die FD-Methode durch die im Jahr 2000 entwickelte LIFDI-Technik [1]. Hierbei wird die gelöste Probe mit Hilfe des Quellenvakuums angesaugt und es erfolgt der direkte Auftrag auf den permanent im Hochvakuum befindlichen FD-Emitter. Gegenüber dem konventionellen Probenauftrag außerhalb des Messgeräts entfällt so die Re-Justage des Emitters nach dem Wiedereinführen in die Ionenquelle. Dies ermöglicht deutlich höhere Ionenausbeuten [2], raumluftfreies Arbeiten und wesentlich bedienungsfreundlichere Messeinrichtungen. Eine besonders interessante Kopplung ist die der LIFDI-Technik mit Flugzeit-Massenspektro-metern, da die TOF-Analysatoren gegenüber scannenden Analysatoren bekanntermaßen Vorteile bei der Detektion schwacher Ionenströme haben. Die daraus resultierenden, nochmals verbesserten Nachweisempfindlichkeiten, hochaufgelöste Messungen mit der Möglichkeit zur Bestimmung von Summenformeln und nicht zuletzt kompakte Geräte-abmessungen machen ein derartiges LIFDI-TOF-MS zu einem interessanten System neben den etablierten Techiken wie ESI- und MALDI-MS. Der besondere Stellenwert innerhalb des Methodenverbunds zur Strukturaufklärung besteht in der Möglichkeit zur Untersuchung wenig polarer Verbindungen, oxidationsempfindlichen und chemisch labilen Systemen. Der Beitrag beschreibt den Einsatz der LIFDI-Technik an einem kommerziellen TOF-MS (GCT der Fa. Micromass) sowie Anwendungsbeispiele aus der industriellen Analytik. Literatur [1] H. B. Linden, European J Mass Spectrom. 10, 459-468 (2004) [2] H. B. Linden, Proceedings of the 49 th Annual Conference on mass Spectrometry and Allied Topics, Chicago 2001, MPA 024

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Poster P36 LC-MALDI on Disposable Prespotted AnchorChip Targets Martin Schuerenberg1, Sven Brand1, Christian Ziegmann2, W. Jabs1, Stephanie Hahner1, and D. Suckau1 1

Bruker Daltonics, Bremen, Germany;2Eppendorf AG, Hamburg, Germany

The coupling of liquid chromatography (LC) with mass spectrometry (MS) is a widely used method for direct analysis of complex protein digest mixtures. To date, LC-MS analysis is performed by single- or two-dimensional separation of digested protein mixtures followed by online electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis. Typical for such online LC-MS analysis are the temporal constraints for further MS/MS precursor ion selection, which enable only the acquisition of MS/MS spectra of the analysis of the eluted peptides. Especially co-eluting peptides are often missed due to the relatively long duty cycle of the ESI-MS for selection of potential peptides for subsequent MS/MS analysis. The use of matrix assisted laser desorption/ionization mass spectrometry (MALDIMS) offline coupled for the analysis of LC separated complex protein mixtures offers significant advantage over ESI since the eluted peptides are directly loaded onto a MALDI target as discrete spots enabling a long-term storage of the samples for subsequent MS and MS/MS analysis without temporal restrictions. In addition, the LC-MALDI approach enables the re-analysis of samples without repeating the chromatographic step. Here we show the use of disposable Prespotted AnchorChip MALDI targets for proteomics applications such as LC-MALDI. These MALDI-targets are equipped with following features: a) Prestructured small hydrophilic patches (“anchors”) on the hydrophobic plastic surface allow the analyte solution to concentrate leading to significantly increased sensitivity. This effect has previously been described for conventional reusable so called “AnchorChip” targets [1,2]. b) Prespotting of matrix and/or calibrants performed under controlled conditions allows convenient reproducible and reliable use for high throughput proteomics. Conventional MALDI targets are reusable, but generally suffer from the well known “memory effect”, i.e., residual analytes which sometimes cannot be removed quantitatively and which obscure the result of the subsequent measurements on the same sample position. Single-use targets are inherently safe with regard to this effect. Another advantage of the disposable Prespotted AnchorChip MALDI targets is the possibility for a cost-efficient archiving of samples. The disposable Prespotted AnchorChip MALDI targets with prespotted -cyano-4hydroxycinnamic acid matrix anchors (800 µm) were used for direct deposition of the LCeluate, followed by a simple collective washing step. The thin layer matrix spots tolerate up to 50% acetonitrile without being dissolved. 100 amol detection sensitivity was achieved on these matrix anchors as they concentrate the analyte from the larger droplets that were deposited. 80% MS/MS sequence coverage was obtained for 100 fmol BSA digest separated in 70 LC-fractions on a Prespotted AnchorChip MALDI-target. The use of these MALDItargets has also been shown to be useful for the analysis of more complex protein digest samples with nano-LC-gradients up to 2 h. [1] M. Schürenberg et al., Anal. Chem. 72 (2000) 3436 – 3442 [2] J. Gobom et al., Anal. Chem. 73 (2001) 434-438

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Poster P37 A quantitative proteomic strategy to study leaf senescence in Arabidopsis thaliana by metabolic labeling and mass spectrometry R. Hebeler1, Kai Reidegeld1, P. P. Dijkwel2, Marcel J. G. Sturre2, H. E. Meyer1 and B. Warscheid1 1

2

Medical Proteom-Center Ruhr-University Bochum, Germany Department Molecular Biology of Plants, Research school GBB, University of Groningen, Netherlands

Arabidopsis thaliana, a small flowering plant, is used as model organism to study the onset of leaf senescence. In general, this process is controlled by leaf age and can be promoted significantly by ethylene within a specific age window [1]. Onset of leaf death (old) mutants of A. thaliana have been studied by senescence associated gene (SAG) expression, ion leakage, and chlorophyll degradation. Advanced leaf senescence has been revealed in old2 plants treated by ethylene, and additionally in old1 and old3 mutants treated by ethylene or air [1]. In this work, an analytical strategy has been developed to provide accurate information on protein expression levels of A. thaliana with normal and altered leaf senescence. Plant tissues were processed under liquid N2 and in the presence of protease inhibitors. After centrifugation of the cell extract, the soluble sample fraction was collected, andThis method avoids any loss of proteins in the course of sample preparation and results in two different fractions, one comprising mainly the membrane and and DNA associated proteins were extracted from the pellet by additional homogenation and centrifugation, the other one containing predominantly [2]. Cytosolic proteins were separated via After two-dimensional gel electrophoresis (2-D PAGE) following the protocol by Klose et al. [3], and subsequently identified by differentially expressed proteins should be cut off the gel, digested with Trypsin and analysed using MAL DI- Mmass sSpectrometry (MS) and SEQUEST™ searches in the NCBI database. To overcome limitations regarding differential protein analysis by 2-D PAGE, the wild type and mutants of Arabidopsis thaliana have been metabolically labeled using 14N/15Nammonium sulphate (98 % purity). Stable isotope incorporation into proteins during plant growth was determined to be 95 %. The linearity of protein quantitation was shown by analyzing protein extracts of five different concentration ratios from metabolically labeled plants. Generally, relative quantitative determination of proteins is based on signal intensities of protonated tryptic peptide pairs detected by nano-high performance liquid chromatography coupled with electrospray ionization quadrupole time of flight mass spectrometry (nHPLC/ESI-qTOF MS). The in-house developed software package Peakardt was employed for advanced quantitative MS data evaluation. [1] [2] [3]

Jing, H.C., et al., Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence. Plant J, 2002. 32(1): p. 51-63. Giavalisco, P., et al., Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis. Electrophoresis, 2003. 24(1-2): p. 207-16. Klose, J. and U. Kobalz, Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis, 1995. 16(6): p. 1034-59.

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Poster P38 Cryodetector-mass spectrometric analysis of plasma proteins enables differentiation of HELLP patients from healthy women. J. Heitner 1,2,*), C. Koy 1,*), R. Woisch 3), M. Kreutzer 1), P. Serrano-Fernandez 1), R. Gohlke 3), T. Reimer 2), and M.O. Glocker 1,*) 1) Proteome Center Rostock, University of Rostock, Rostock, Germany. 2) Department of Obstetrics and Gynecology, University of Rostock, Germany. 3) GSG Mess- und Analysengeräte GmbH, Bruchsal, Germany. The Hemolysis, Elevated Liver enzymes, and Low Platelets syndrome (HELLP syndrome) is a serious, life-threatening variant of severe preeclampsia in pregnant women. Up to now, neither reliable preclinical recognition nor effective prevention measures for HELLP patients are available. The pathogenesis of HELLP syndrome is unknown, therefore a causal therapy is impossible [1,2]. Our goal is to develop a cryodetector mass spectrometry-based screening method [3] that enables the rapid and reliable detection of HELLP syndrome. For developing the assay, plasma protein abundances from patients suffering from HELLP were profiled before and after delivery and compared to healthy pregnant control individuals, using a time-of-flight mass spectrometer equipped with a cryodetector system. The spectra were well reproducible when acquisition conditions were kept constant and the overall appearance of the profiles was well comparable. Peak areas of ten selected ion signals (9.5, 11.8, 14.0, 28.1, 43.4, 50.5, 60.1, 66.6, 74.5, and 79.8 kDa) from each spectrum were subjected to statistical analysis. Significant differences in ion intensities between the groups could be measured even without sample fractionation. The most striking difference between most of the spectra from HELLP patients and spectra from non-HELLP samples was the presence/absence of the 11.8 kDa ion signal (p-value: 0.0000365), presumably belonging to serum amyloid A (SAA). Other significant differences in ion intensities between HELLP and control samples were observed in the peak areas of the 14.0 kDa, the 28.1 kDa, the 50.5 kDa, and the 74.5 kDa ion signals. Using the cryodetector mass spectrometry data for sorting plasma samples into either the HELLP group or the non-HELLP group, a sensitivity of 81.25% and a specificity of 95.45% were achieved. In order to verify the mass spectrometrically determined profiling results, plasma samples from patients suffering from HELLP as well as from controls were subjected to 2D-gel electrophoresis. One of the remarkable findings in the comparison of gels was that in case of HELLP expression of SAA was differentiating the two groups, as advanced by the results of the cryodetector analysis. The protein spots were present in five out of six HELLP-patients for which 2D-gel analyses were performed. Literature [1] Weinstein L. (1982). Syndrome of hemolysis, elevated liver enzymes, and low platelet count: A severe consequence of hypertension in pregnancy. Am. J. Obstet. Gynecol. 142, 159-167. [2] Rath W., Faridi A., Dudenhausen J.W. (2000). HELLP syndrome. J. Perinat. Med. 28: 249-260. [3] Christ P., Rutzinger S., Koy C., Seidel W., Uchaikin S., Glocker M.O., Pröbst F. (2004). High detection sensitivity achieved with cryogenic detectors in combination with matrix-assisted laser desorption / ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Eur. J. Mass Spectrom. 10: 469-476.

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Poster P39 Salt-free Iin-gel digestion of proteins in salt-free media using by covalently modified trypsin. s in 10 % Acetonitrile Henrik Thomas 1, Marek Sebela 2 und Andrej Shevchenko 1 1

Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany. 2 Palacky University, Department of Biochemistry, Slechtitelu 11, CZ-783 71 Olomouc, Czech Republic.

We demonstrated that covalently mModified trypsins, such as like methylated porcine trypsin (Promega) and a new rafinose modified bovine trypsin (sugar modified lysins) can efficiently 2 are able to digest pProteins in at neutral pH, without any buffer salts, but with 10 % acetonitrile with no pH-buffering component.instead. MALDI TOF-MS spectra of high quality have beenwere acquieredacquired using enhanced dried-droplet probe preparation on AnchorChipTM targets (Thomas et al, RCM 18, 923 (2004))[1]. NoThe method allows skipping the desalting step in sample preparation for MALDI-MS and nanoESI-MS is required. . No chemicals, which may influence mass spectrometric analysis, have to be added. The organic solvent at this concentration works as mild softly denaturating agent for the enzyme (like urea or ammonium bicarbonate which are used normaly) and increases the recovery of peptide digestion products and has a positive influence on digestion. It alsodecreases the adsorption of prevents peptides from adsorption by the wallon the surface of polyethylene tubes., if the supernatant is used without extraction. Comparison of peptide mass maps acquired from in-gel digests of protein standards using salt-free and conventional recipes suggested that cleavage specificity and completeness were not affectedSequence specifity is not influenced. Digestion can be carried done at elevated higher temperatures (up to 55oC ) [2], while protein digestion by . uUnmodified trypsin does not work in this way.under these conditions is not efficient. Literatur [1] Thomas H, Havlis J, Peychl J, Shevchenko A. Rapid Communications in Mass Spectrometry 18 (9): 923930 (2004) [2] Havlis J, Thomas H, Sebela M, Shevchenko A. Analytical Chemistry (Washington) 75 (6): 1300-1306 (2003)

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Poster P40 Combinatorial Assembly of Heterometallic Double Helicates H. Herschbach 1, E. Leize 1, A. Van Dorsselaer 1, A. Marquis 2, J. M. Lehn 2 1

Laboratoire de Spectrométrie de Masse Bio-Organique, Université Louis Pasteur, 25, rue Becquerel, F-67087 Strasbourg. 2 Institut de Science et d´Ingénierie Supramoléculaires, 8, allée Gaspard Monge, F-67083 Strasbourg

Tritopic ligands containing bidentate (B) and / or tridentate (T) sites (Fig. 1) were synthesized where B and T denote bi- and ter-pyridine-like binding sites. Double helicate structures can be obtained by complexation of metals like copper, zinc or iron [1-3]. Cu(I) prefers to be tetracoordinated and will thus occupy sites between two bipyridine (B) sites in the double helicate (Fig. 2). Cu(II) is pentacoordinated (appropriate binding geometry between B and T), Zn is hexacoordinated (between T and T).

N

N

N

O

N

N

BBT BBT

N

N

O

BBT

BBT TBB

N N

N

O

N

N

TBT TBT

N O

N

N

BBT TBT

TBT

Fig. 1: Structures of two examples of tritopic ligands: bipyridine-bipyridine-terpyridine strand (BBT) and terpyridine-bipyridineterpyridine strand (TBT).

Tetra - -

CuI

Penta -

CuII

Hexa -

ZnII coordinated

Fig. 2: Coordination geometry of Cu(I), Cu(II) and Zn(II) and the four possible helicates in the mixture of the three metals and BBT and TBT strands.

In the present work, we analysed the species distribution arising when mixtures of up to five tritopic polypyridine ligands (BBB, BBT, BTB, TBT, TTT) are added to solutions containing Zn(II), Cu(I) and Cu(II) cations. ESI-MS allowed characterisation of the complexes generated by the 16 possible ligand-metal combinations. It is possible to control ligand pairing by the specific coordination preferences of the metals. This suggests the possibility of a simple analogue of the polymerase chain reaction in which simple chelate entities may be assembled on a single strand helicand-metal complex. References [1] Smith, V.C.M., Lehn, J.M., J. Chem. Soc., Chem. Commun. 1996, 2733. [2] Albrecht, M. Chem. Rev. 2001, 101, 3457. [3] Hasenknopf, B., Lehn, J.M., Baum G., Fenske D., Proc. Nat. Acad. Sci. U.S., 1996, 93, 1397.

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Poster P41 Analyse von Daten der bildgebenden MS durch visuelle Verfahren A. Hester, W. Bouschen, A. Leisner, K. Maaß, C. Paschke, B. Spengler Institut für Anorganische und Analytische Chemie, Universität Gießen, 35392 Gießen Die Untersuchung von biologischem Gewebe mit SMALDI-MS (Scanning Microprobe MALDI Mass Spectrometry) erzeugt sehr große zu verarbeitende Datenmengen. So ergibt eine 100µm*100µm Probenfläche, die mit 1µm Schrittweite in 10 Durchgängen (Schichten) abgerastert wird, 100 000 Massenspektren. Um eine solche Informationsmenge in vertretbarer Zeit auswerten zu können ist eine hochgradig automatisierte Datenverarbeitung unumgänglich. Dies erfordert, insbesondere wegen der Überlagerung diverser physikochemischer Prozesse, neben der bloßen visuellen Darstellung der Messdaten eine umfangreiche Vorverarbeitung, so dass sich Aussagen über biologische und medizinische Zusammenhänge aus den Daten extrahieren lassen. Schließlich ist es erforderlich, dass ein solches Hilfsmittel sowohl die Verwaltung, als auch den Rückgriff aus analytischen Abbildungen auf die zugrundeliegenden Rohdaten bzw. auf Daten einzelner Verarbeitungsschritte übernimmt. Das Poster stellt das Konzept und die wesentlichen Algorithmen einer Software vor, mit der Rohdaten erfasst, gespeichert und verwaltet werden. Die Spektren werden auf relevante Massensignale (Peaks) hin untersucht. Diese Peaks werden markiert und kategorisiert (Form, Größe, Höhe sowie signifikante Punkte werden bestimmt). Anschließend wird aus den markierten Peaks der gerasterten Probenfläche eine vierdimensionale Matrix erstellt. Aus dieser wird ein Histogramm errechnet, das den Informationsgehalt als Funktion der Masse quantifiziert und es somit dem Anwender (als Alternative zu einer automatischen Auswahl) ermöglicht, interessante Peaks bzw. Massenbereiche auszuwählen. Für diese Massenbereiche werden dann grafische Darstellungen erzeugt. Zur Klärung biologischer Fragestellungen lassen sich die Darstellungen unterschiedlicher Massen miteinander vergleichen, indem sie überlagert und rechnerisch verknüpft werden (z.B. Differenzbild). Ebenso können Bilder einer Probe, die anderen Verfahren, etwa der optischen Mikroskopie, entstammen, mit den massenspektrometrisch gewonnenen Bildern der Probe kombiniert werden. Die Strukturerkennung (Segmentierung) unterstützt den Anwender sowohl bei seiner Arbeit, als auch bei der Vermessung und Quantifizierung der Bilddaten. Ein wesentlicher Punkt, der eine spezielle Bildanalysesoftware notwendig macht, da hier professionelle Bildverarbeitungsapplikationen versagen, ist der Rückgriff vom erstellten Bild auf die Rohdaten, d.h. auf einzelne Massenspektren. Die Bilddarstellung der Peakdaten dient so als Landkarte. Die Auswahl eines Pixels führt sodann zur Darstellung des zugehörigen Massenspektrums. Das Projekt wurde gefördert vom Bundesministerium für Bildung und Forschung, Aktenzeichen 0312834A und von der Thermo Electron GmbH, Bremen.

148

Poster P42 Wechselwirkung von UV-Strahlung mit der Haut – MALDI und ESR-tomographische Messungen Hochkirch, U. 1, Herrmann, W. 2, Stößer, R. 1, Borchert, H.H. 2 und Linscheid, M. 1 1

Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Str.2, 12489 Berlin. 2 Institut für Pharmazie, Freie Universität Berlin, Kelchstr.31, 12169 Berlin.

Die Haut ist das größte Körperorgan des Menschen und stellt gleichzeitig die äußere Barriere gegen Umwelteinflüsse wie die Strahlung der Sonne dar. Seit langem werden ihrem ultravioletten Spektrum, neben diversen positiven Effekten wie Vitamin-D-Bildung und Hautbräunung, auch unerwünschte Wirkungen auf die Haut zugeschrieben. Dazu zählen die durch den kurzwelligen Bereich (UVB: 290-315nm) verursachte lichtbedingte Hautalterung und die direkte Schädigung von DNA. Der längerwellige UVA-Anteil bedingt dagegen durch die Anregung photosensibilisierender Moleküle die Bildung von radikalischen Spezies, die ihrerseits zu Schäden an Erbgut und Zellmembranen führen [1, 2]. Mit der ESR (Elektronenspinresonanz)-Tomographie existiert eine Meßmethode, Radikale in den einzelnen Hautschichten direkt nachzuweisen. Da durch Licht erzeugte Radikale bei Raumtemperatur sehr kurzlebig und somit ihre stationären Konzentrationen für die ESRTomographie nicht ausreichend sind, wird in dieser Arbeit ein Spinlabel in Hautbiopsien gebracht, um dessen Reaktion auf die UV-Einwirkung zu untersuchen. Mit Hilfe von Versuchen an Gelatineschichten, die häufig als Modell für Körperteile dienen, konnte die Eindringtiefe von UVA- und UVB-Strahlung durch ihre Wirkung auf das Spinlabel ortsaufgelöst dargestell werden. Ein Verfahren zur quantitativen Auswertung solcher ESRTomogramme wird derzeit entwickelt.

ESR-Tomogramm einer Hautbiopsie nach Tauchen in wasserlöslichem Spinlabel. Die unterschiedliche Hydrophilität der einzelnen Hautschichten ist gut erkennbar. Rechts: Standard zur Quantifizierung

Parallel dazu werden ebenso behandelte Hautproben mit MALDI-MS untersucht. Anregung mit einem UV-Laser führt zu einer teilweisen Umwandlung des Spinlabels in das entsprechende Hydroxylamin im Massenspektrometer selbst, so dass eine Aussage über die ursprünglich vorhandene Radikalkonzentration nur indirekt möglich ist. Die Ionisierung soll deshalb mit einem IR-Laser erfolgen, bei der ggf. sogar auf den Einsatz einer Matrix verzichtet werden kann. Literatur [1] Altmeyer, P.; Skin Cancer and UV Radiation; Springer Berlin; 1997 [2] Halliwell,B., Gutteridge, J.M.C.; Free Radicals in Biology and Medicine; Oxford University Press; 2001

149

Poster P43 Thin-layer chromatography combined with diode laser desorption/atmospheric pressure chemical ionization mass spectrometry Song Peng 1, Norman Ahlmann 1, Kerstin Kunze 1, Walter Nigge 1, Michael Edler 1, Thorsten Hoffmann 2, and Joachim Franzke 1 1

ISAS – Institute for Analytical Sciences, Bunsen-Kirchhoff-Strasse 11, 44139 Dortmund, Germany 2

Institute of Inorganic Chemistry and Analytical Chemistry, Johannes GutenbergUniversity, Duesbergweg 10-14, 55128 Mainz, Germany

The desorption of an analyte by a continuous wave diode laser from a porous surface of a thin layer plate covered with a graphite suspension is presented. The thermally desorbed analyte molecules are ionized in the gas phase by a corona discharge at atmospheric pressure. Therefore, both essential processes – the desorption and the ionization of analyte molecules, which are often performed in one step - are separated. The target preparation is easy and fast since no additional extraction process is required. The mass spectrometric background signal was mostly limited to the low mass range showing no interference with typical compounds of interest. In this study, the calmative and antihypertensive drug reserpine was chosen as model analyte, which is often used for tuning mass spectrometers in our laboratory. No fragmentation was observed because of efficient collisional cooling under atmospheric pressure. The influence of diode laser power and the composition of graphite suspension was investigated, and a primary optimization was performed.

150

Poster P44 NanoLC MALDI MS und MS/MS – Methodenentwicklung für die Identifizierung von MHC Klasse I gebundenen tumor-assoziierten Peptidantigenen Sandra Hofmann1, Matthias Glückmann2, Sandra Kausche3, Andrea Schmidt1, Wolfgang Herr3, Michael Karas1 1

Institut für Pharmazeutische Chemie, Johann Wolfgang Goethe Universität, Marie-Curie-Str. 9, 60439 Frankfurt, Deutschland, 2 Applied Biosystems, Frankfurter Str. 129B, 64293 Darmstadt, Deutschland, 3 III. Medizinische Klinik, Hämatologie und Onkologie, Johannes Gutenberg-Universität Mainz, Langenbeckstr. 1, 55101 Mainz, Deutschland

ESI MS ist bisher die Methode der Wahl zur Detektion von LC basierten Peptidanalysen, während MALDI TOF hauptsächlich zur „peptide mass fingerprint“ Analyse (PMF) angewendet wird. Ein Vorteil der Analyse mittels LC MALDI Kopplung ist, dass die LCFraktionierung und die MS bzw. MS/MS Analyse getrennt sind. Daher können z.B. die Precursor manuell festgelegt werden und zusätzlich können Proben auf den MALDI Tellern aufbewahrt und reanalysiert werden. NanoLC spotting wurde mit nachfolgender MALDI TOF bzw. TOF/TOF Analyse als Methode evaluiert. Dabei wurde besonders auf Sensitivität und Reproduzierbarkeit geachtet. Angewendet wurde die Kopplung auf die Identifizierung von Epitopen von Haupthistokompatibilitätskomplex (MHC) Klasse I assoziierten zytotoxischen TLymphozyten (CTL). MHC Klasse I Moleküle wurden aus einer Zelllinie eines menschlichen klarzelligen Nierentumors immunchromatographisch aufgereinigt und daraus die MHC assoziierten Peptide extrahiert. In einem solchen Extrakt befinden sich normalerweise etwa 10e4 unterschiedliche Peptidliganden, die überwiegend aus physiologisch exprimierten Proteinen und in der Minderheit auch aus tumorspezifisch exprimierten Proteinen prozessiert werden. Die Peptidliganden wurden zunächst mittels RPHPLC fraktioniert und schließlich via RP nanoLC in Kombination mit online spotting auf MALDI Teller analysiert. Nach MALDI TOF/TOF Analyse wurden die erhaltenen MS/MS Spektren einer Datenbanksuche unterworfen sowie manuell interpretiert. Mit dieser Methode konnte eine Vielzahl an MHC gebundenen Tumorpeptiden identifiziert werden. Identifizierung und Charakterisierung von MHC Klasse I assoziierten Peptiden, die bei Tumoren, viralen Infektionen oder Autoimmunerkrankungen neu generiert werden, sind ein wichtiger Schritt zum Verständnis der Interaktionen zwischen dem T-zellulären Immunsystem und antigenen Peptiden. Dieses Verständnis ist die unmittelbare Voraussetzung für eine verbesserte Spezifität und Effektivität von immunotherapeutischen Strategien, insbesondere mittels gezielter Impfungen oder adoptivem CTL-Transfer.

151

Poster P45 Determination of Iodine Species in the Coastal Atmosphere using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) H.Chen1, R.Brandt2, R.Bandur2, N.Jakubowski2, K.G.Heumann1, T.Hoffmann1 1. Institute of Inorganic Chemistry & Analytical Chemistry, Johannes Gutenberg – University, Duesbergweg 10-14, D-55128, Mainz, Germany. 2. Institute for Analytical Sciences (ISAS), Bunsen-Kirchhoff-Str.11, D-44139, Dortmund, Germany. It has been demonstrated that iodine does have an important influence on atmospheric chemistry, especially the formation of new particles and the enrichment of iodine in marine aerosols. It has been pointed out that the most probable chemical species involved in the production or growth of these particles are iodine oxides, produced photochemically from biogenic halocarbon emissions and/or iodine emission from the sea surface. Recent chamber and model studies have confirmed those particle formations from I2 or CH2I2 in the presence of ozone and UV light. However, the iodine chemistry from gaseous to particulate phase in the coastal atmosphere and the chemical nature of the condensing iodine species are still not understood. Therefore, sampling, identification and quantification of those iodine species have become necessary for the understanding of atmospheric iodine chemistry. A denuder sampling technique has been developed to characterise potential precursor compounds of coastal particle formation processes, such as molecular iodine in the gas phase. Starch, TMAH and TBAH coated denuders are tested to collect I2 at the inner surface, followed by a TMAH extraction and ICP/MS determination, adding tellurium as an internal standard. A Water/TMAH-Extraction-ICP/MS method has been developed for the accurate and sensitive determination of iodine species in tropospheric aerosol particles. The samples are collected on cellulose-nitrate filters using conventional filter holders or on cellulose nitrate/tedlar-foils using a 5-stage Berner impactor for size-segregated particle analysis. The water soluble species as IO3- and I- are separated by anion exchanging process after water extraction. Non-water soluble species including iodine oxide and organic iodine are extracted by tetramethylammonium hydroxide (TMAH). Afterwards the triple samples are analysed by ICP/MS. The detection limit for particulate iodine is determined to be 0.10~0.20 ng·m-3 for sampling volumes of 40~100 m3. The developed methods have been used in 2 field measurements in May 2002 and September 2003, at and around the Mace Head atmospheric research station located at the west coast of Ireland. Results from the measurement campaigns and the laboratory chamber, field seaweed-chamber experiments will be presented and discussed within this contribution.

References [1] Kolb, C.E. Nature, 2002, 417, 597-598. [2] O‘Dowd, C.D.; Jimenez, J.L. et.al. Nature, 2002,417, 632-636. [3] Rädinger, G.; Heumann, K.G. Anal.Chem., 1998, 70, 2221-2224 [4] Wimschneider, A.; Heumann, K.G. Fresenius J. Anal. Chem,. 1995, 353, 191-196

152

Poster P46 Untersuchung von RNA / DNA Dimeren und Minor Groove Binding Ligands mittels nano-ESI-MS Corina Hunger und Michael Karas Institut für Pharmazeutische Chemie, Johann-Wolfgang-Goethe Universität Frankfurt a.M., Marie-Curie-Str. 9-11, 60439 Frankfurt Das Interesse an Oligonukleotiden als „drug target“ nimmt immer weiter zu. Dabei werden sowohl die Oligonukleotide selbst (RNAi, RNAantisense) als mögliche Kandidaten verwendet, ebenso kommen auch RNA und DNA direkt als Wirkort für mögliche Medikamente in Frage. Die Untersuchung solcher Komplexe mittels Massenspektrometrie ist von großem Vorteil, da diese eine schnelle Analyse ermöglicht und auch die Bindungstöchiometrie sofort ersichtlich ist. Die Spezifität von RNAsense und RNAantisense konnte bereits mittels nano-ESI nachgewiesen werden[1]. Dabei wurden allerdings nur solche Fälle betrachtet, die entweder 12 oder mehr, bzw. 2 oder weniger komplementäre Basenpaarungen aufwiesen. In der vorliegenden Untersuchungen wurden daher verschiedene Modellsysteme sowohl für RNA als auch für DNA ausgewählt, die zwischen 0 und 12 Basenpaarungen besitzen, bzw. deren Bindungsenergien zwischen 0 und 25 kcal/mol betragen. Mit diesen Systemen konnte gezeigt werden, dass nur Komplexe mit Bindungsenergien über 10 kcal/mol mittels nanoESI als spezifisches Dimer detektiert werden können. Unspezifische Komplexe im Massenspektrum konnten durch einfache Verdünnungsexperimente eliminiert werden. Generell waren RNA-Dimere im Vergleich zu den entsprechenden DNA-Dimeren stabiler, was auf die höheren Bindungsenergien zurückzuführen ist. Diese Spezifität der Komplexe konnte sowohl für RNA- und DNA- Homodimere, als auch für die entsprechenden Heterodimere nachgewiesen werden. Eine interessante Gruppe von DNA-bindenden Liganden sind die Minor Groove Binding Ligands (MGBL). Dabei handelt es sich um Polyamide mit einem N-Methylpyrrol- oder NMethylimdiazolgerüst, die an die Minor Groove der DNA binden und eine sichelförmige Struktur aufweisen. Die Bindung an die DNA ist dabei sequenzspezifisch, wobei eine hohe Affinität gegenüber AT-reichen Regionen besteht. MGBL werden beispielsweise als Antitumormedikamente oder bei der HIV-Behandlung (verminderte Transfektion) eingesetzt. Durch die Form und Ladung der Liganden besitzen die Komplexe eine spezifische Bindungsstöchiometrie, d.h. es können entweder ein oder zwei Liganden in der Minor Groove einer DNA binden[2],[3]. Mittels nano-ESI konnte eine Affiniät der MGBL für AT-reiche Sequenzen gezeigt und die Bindungsstöchiometrie bestimmt werden. Weiterhin zeigte sich, daß die Bindung an die DNA-Minor Groove spezifisch ist, während die Minor Groove der RNA zu flach ist für die Bindung mit MGBL. Damit ist es möglich, die Bindungstöchiometrie und die Spezifität neu synthetisierter Liganden zu testen. Literatur [1] Hunger, C. and M. Karas,MPR347,51st ASMS Conference. 2003. Montreal. [2] Rosu, F., et al.,. Nucleic Acids Res, 2002. 30(16): p. e82. [3] Gupta, R., et al., J Am Soc Mass Spectrom, 2004. 15(10): p. 1382-91.

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Poster P47 Proteome Analysis of Escherichia coli using High-Performance Liquid Chromatography and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Christian Ihling & Andrea Sinz Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, D-04103 Leipzig, Germany The basic problem of complexity poses a significant challenge for proteomics studies. To date, 2-DE (two-dimensional gel electrophoresis) followed by enzymatic in-gel digestion of the peptides, and subsequent identification by mass spectrometry is the most commonly used method to analyze complex protein mixtures. However, 2-DE is a slow and laborintensive technique, which is not able to resolve all proteins of a proteome. To overcome these limitations gel-free approaches are developed based on high-performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). The high resolution and excellent mass accuracy of FTICRMS provides a basis for simultaneous analysis of numerous compounds [1]. In the present study, a smallprotein subfraction of an E. coli cell lysate was prepared by size-exclusion chromatography and proteins were analyzed using C4 reversed phase HPLC for pre-separation followed by C18 reversed phase nano-HPLC/nano-ESI-FTICRMS for analysis of the peptide mixtures after tryptic digestion of the protein fractions [2]. We identified 231 proteins and thus demonstrated that a combination of two reversed phase separation steps - one on the protein and one on the peptide level - in combination with high-resolution FTICR mass spectrometry has the potential to become a powerful method for global proteomics studies. [1] Comisarov, M.B., Marshall, A.G. (1974) Chem. Phys. Lett. 25, 282-283. [2] Ihling, C., Sinz, A. Proteomics, in press.

154

Poster P48 Untersuchungen zum Mechanismus der ISD-Fragmentierung am Beispiel der oxidierten Insulin B-Kette T. Jaskolla, M. Karas Institut für Pharmazeutische Chemie der Johann Wolfgang Goethe Universität Frankfurt am Main, Marie-Curie-Str. 9-11, 60439 Frankfurt [email protected] Die schnelle metastabile Fragmentierung (ISD: In Source Decay) tritt bei MALDI innerhalb von wenigen Nanosekunden nach Laserbeschuss noch vor Anlegen der Beschleunigungsspannung in delayed-extraction TOF MS-Systemen auf. Das ISDFragmentierungsmuster unterscheidet sich signifikant von PSD- bzw. CID Massenspektren. Der Einfluss der Aminosäuresequenz bzw. der Aminosäureseitenketten auf die bei der ISDFragmentierung auftretenden verschiedenen Fragmentionenserien ist Gegenstand dieser Analyse. Die Intensitäten der einzelnen Zerfallsprodukte werden in Abhängigkeit ihres Fragmentierungstypus analysiert und die beobachteten Intensitätsunterschiede für die verschiedenen Fragmentierungswege unter Berücksichtigung der Einflüsse der Seitenketten diskutiert. Weiterhin erfolgt eine Analyse der Fragmentionen-Intensitäten aller Zerfallsserien in Abhängigkeit des Polaritätsmodus und damit eine Betrachtung der Fragment-Protonierungstendenz bzw. der -gasphasenbasizität. Da die Gasphasenbasizitäten bisher nur experimenteller Ermittlung zugänglich sind, erfolgt die Korrelation anhand der berechenbaren lösungsbezogenen pKi-Werte der ISD-Fragmente. Es wurde eine ausgeprägte Übereinstimmung der Signalintensitäten mit dem Fragment-pKi gefunden, zum Beispiel konnte gezeigt werden, daß z-Fragmente durch Verlust ihrer N-terminalen Aminofunktion saurer werden und sich somit leichter im Negativ-Modus detektieren lassen. Anhand der Analyse gemittelter Fragmentionen-Intensitäten war es u. a. möglich, den Fragmentierungsmechanismus für die a-Ionenserie der ISD-Fragmentierung näher zu charakterisieren. Auch kann geschlossen werden, daß bei b-Ionen eine Zyklisierung unter Ausbildung azider Protonen erfolgen muss. Es wurde weiterhin festgestellt, daß vor Cysteinsäuren sehr intensive c-Fragmente auftreten; dafür wurde ein theoretisches Modell entwickelt, das anschließend anhand von weiteren im Massenspektrum detektierbaren Signalen verifiziert werden konnte. Die detaillierte Analyse zeigte eine neue Fragmentionenserie, die auf NH-Abspaltung aus der c-Serie zurückzuführen ist. Es wurde festgestellt, daß vor ox. Cysteinen und vor Argininen hohe y-Fragmentintensitäten auftreten; dieses kann durch eine Stabilisierung über eine Zyklisierungsreaktion erklärt werden. Auch konnte eine zur z-Fragmentierung konkurrierende Abspaltung von MethylenimmoniumIonen aus y-Fragmenten mit N-terminalem Glycin nachgewiesen werden.

155

Poster P49 Differential proteome analysis of tonsil material from children with chronic tonsillitis in comparison with hyperplasia in order to identify diagnostic marker proteins Tino Just 1, Evariste Gafumbegete 2, Jan Gramberg 1,3, Ines Prüfer 3, Bruno Ringel 3, Hans Wilhelm Pau 1 and Michael O. Glocker 3 1

Department of Otorhinolaryngology, Head and Neck Surgery, University of Rostock Department for Pathology, University of Rostock 3 Proteome Center Rostock, University of Rostock 2

Differential proteome analysis [1] was used to compare protein pattern from fresh-frozen tonsils derived from children with recurrent and chronic tonsillitis, and tonsil hyperplasia, respectively. In order to find disease associated marker proteins, protein pattern of both entities were compared. Sixty-eight corresponding pairs of tonsil from children, aged between 2 and 11, were removed surgically between May 2003 and January 2004. Thirty-three protein probes were isolated (24 from chronic tonsillitis and 9 from hyperplasia). Two-dimensional (2D) gel electrophoresis of the soluble proteins was performed. All gels were stained with colloidal Coomassie Brillant Blue G-250. Differential computer-based 2D-gel analysis was carried out using two 2D reference gels of each entity. The selected protein spots were excised manually and digested with trypsin using an Investigator ProGest system. In order to identify tonsil proteins, mass spectrometric peptide mass fingerprinting was performed by MALDI-TOF-MS and database search with the Proteomic Tool at the Swiss-Prot Database, respectively. 600 Protein spots were visualized routinely on each gel using Coomassie staining. A strong similarity in abundance of protein spots within each group was apparent. Only those differences that persisted in the rigorous differential analysis were addressed as differentially expressed, resulting in rather small differences in protein patterns between tonsillitis and hyperplasia. Eighty-one protein spots were selected for mass spectrometry. Clear abundance differences between the two groups were found for heat shock protein 27 (HSP27) and UMP-CMP kinase. The spots of both proteins were more intensive in chronic tonsillitis. HSP27 ELISA (EKS-500) analyses are performed to verify the different abundance of HSP27 with extracts from 35 frozen tonsils. HSP27 is expressed in many tissues following stress including inflammation, but also in cancer and lymphoma [2, 3]. In further investigations we will evaluate the applicability of HSP27 as diagnostic marker for tonsil disease. References 1

2 3

Sinz A, Bantscheff M, Mikkat S, Ringel B, Drynda S, Kekow J, Thiesen HJ, Glocker MO: Mass spectrometric proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis. Electrophoresis 2002;23:3445-56. Hsu PL, Hsu SM: Abundance of heat shock proteins (hsp89, hsp60, and hsp27) in malignant cells of Hodgkin's disease. Cancer Res 1998;58:5507-13. Sarto C, Valsecchi C, Magni F, Tremolada L, Arizzi C, Cordani N, Casellato S, Doro G, Favini P, Perego RA, Raimondo F, Ferrero S, Mocarelli P, Galli-Kienle M: Expression of heat shock protein 27 in human renal cell carcinoma. Proteomics 2004;4:2252-60.

156

Poster P50 Probing Laminin Self-Interaction by Chemical Cross-Linking and ESI-FTICR Mass Spectrometry Stefan Kalkhof 1, Sebastian Haehn 2, Christian Ihling 1, Neil Smyth 2, and Andrea Sinz 1 1

Biotechnological-Biomedical Center, University of Leipzig, Linnéstr. 3, D-04103 Leipzig, Germany 2 Center for Biochemistry and Center for Molecular Medicine, Faculty of Medicine, University of Cologne, Joseph-Stelzmann-Strasse 52, Cologne D-50931, Germany

We are employing chemical cross-linking [1] in combination with high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain structural information about the self-interacting regions of the N-terminal (LN) domains of laminin [2]. This heterotrimeric glycoprotein is the main non-collagenous component of basement membrane and controls cellular activities such as adhesion or migration, differentiation and polarity, proliferation or apotosis. Laminin is essential for formation of basement membranes through interactions with itself and other components [3]. LN domains (50-60 kDa) were cross-linked with a number of homobifunctional, isotope-labeled, amine-reactive cross-linkers, such as sulfo-DSG (disuccinimidyl glutarate), sulfo-DSA (disuccinimidyl adipate) and BS3 (bis (sulfosuccinimidyl) suberate), bridging distances between ~7 and 12 Å. The extent of cross-linking was monitored by onedimensional gel electrophoresis (SDS-PAGE) and the cross-linked products were cut out and digested in gel. To obtain fragments of appropriate mass (m/z 500-4000 Da) for analysis we used a variety of proteases, such as trypsin, AspN, or LysC as well as combinations of these enzymes. The resulting peptide mixtures were separated by nano-HPLC on reversedphase (C-18) capillary columns (inner diameter 75 µm) applying water-acetonitrile gradients with flow rates of 200 nl/min. The nano-HPLC system is directly coupled to the FTICR mass spectrometer equipped with nano-ESI source. The mass spectrometric data were analyzed using customized software; mass spectra of cross-linked and non-crosslinked peptide mixtures being compared to each other, and signals occurring exclusively in mass spectra of the former were considered as possible cross-linked products. The excellent resolution and accuracy of FTICRMS enhances the confidence in our identification of the cross linked products, which are then used to gain information on the interacting surfaces of the laminin LN domains. [1] [2] [3]

Sinz A, Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes, J Mass Spectrom 38, 1225-1237 (2003) Odenthal U; Haehn S, Tunggal P, Merkl B, Schomburg D, Frie C, Paulsson M, Smyth N, Molecular analysis of laminin N-terminal domains mediating self-interactions, J Biol Chem 279, 44504-12, (2004) Aumailley M, Smyth N, The role of laminins in basement membrane function, J Anat 193, 1-21 (1998)

157

Poster P51 Untersuchung der Solvolyse von tert.-Butylhalogeniden mittels ESI-MS. Björn Klein 1, Francesco Fabbretti 1 und Jürgen O. Metzger 1 1

Institut für Reine und Angewandte Chemie, Universität Oldenburg, Postfach 2503, 26111 Oldenburg Nucleophile Substitutionen und ionische -Eliminierungen sind präparativ sehr wichtige Reaktionen. Hierbei wird zwischen zwei Reaktionsmechanismen unterschieden. Die SN1-Reaktion führt über ein Carbokation als Intermediat zum Produkt, während die SN2-Reaktion aus den Substraten direkt ohne Zwischenstufe zum Produkt führt. Dies gilt ebenfalls für E1 und E2-Reaktionen. Die Stabilitätsreihe der Carbokationen tert. > sec. > prim., die Reihenfolge der Nucleofugie der Abgangsgruppen und der Einfluss der Ionisierungsstärke des Lösungsmittels ist grundlegendes Lehrbuchwissen. Allerdings gibt es keine einfache und allgemeine Methode, die Carbokationen R+ als reaktive Zwischenstufe und die dazugehörigen Anionen X- direkt nachzuweisen sowie deren Bildung direkt zu verfolgen. Wir untersuchten mittels ESI-MS den zeitlichen Verlauf der Solvolyse von tert.Butylhalogeniden in Acetonitril. tBu-X

tBu

+ X

(I)

X = I, Br, Cl Abbildung 1: Reaktionsschema der Ionenbildung

Das tBu-Kation und die entsprechenden Anionen konnten eindeutig nachgewiesen werden. Entsprechend der wohlbekannten Reihe der Nucleofugie der Abgangsgruppen Cl < Br < I stieg die Geschwindigkeit der Bildung der Ionen und ihre relative Intensität an. Weiterhin wurde die Abhängigkeit der Intensität des Signals des tBu-Kations und der Halogenidionen von der Konzentration des Substrats untersucht.

158

Poster P52 ESI-MS Investigation of the Palladium-Catalyzed Allylic Substitution Reactions Larisa Knaack and Jürgen O. Metzger Institute of Pure and Applied Chemistry, University of Oldenburg, P. O. Box 2503, D26111 Oldenburg, Germany Electrospray ionization mass spectrometry (ESI-MS) allows the observation of intermediates involved in catalytic reactions. Palladium-catalyzed reactions of allylic compounds via the formation of -allylpalladium complexes offer many synthetically useful methods.[1] The first step of the catalytic cycle is formation of Pd-allyl complex 1 and the second step is nucleophilic addition to the allyl system, to give product 2 (Scheme 1).[2] +

PdL2

100

1229.1

+ Nu R

R

Ph

OAc PdL2

R

PdL2 Ph 1230.9

R

2

1228.2

+

+ Nu-

1231.9

AcO-

PdL2

R

PdL2

1233.8

R

1077.1

1 1075.1

1081.0

1225.3

1234.8

0 1060

Scheme 1. Pd-catalyzed allylic substitution reaction. R = H, Ph; L = ligand PPh3, PP; Nu- = -CEt(CO2Et)2.

1080

1100

1120

1140

1160

1180

m/z

1200

1220

1240

1260

Fig. 1. Positive-mode ESI-MS spectra of the reacting solution of the allyl acetate and diethyl ethylmalonate in presence of the Pd catalyst.

The allylation of diethyl ethylmalonate with allyl acetate was studied by ESI-MS. The data being directly acquired from the reaction mixture. The expected signals of Pd precatalyst and Pd-bond allyl intermediate with the characteristic isotope distribution for palladium were detected in positive-mode ESI-MS spectra (Fig. 1). Literature [1] J. Tsuji, Palladium Reagents and Catalysts-New Perspectives for the 21st Century, 2nd ed, John Wiley&Sons Ltd, 2004, pp. 431-517. [2] C. Markert, A. Pfaltz, Angew. Chem., Int. Ed. 2004, 43, 2498.

159

Poster P53 Therapeutisches Drug Monitoring (TDM): Quantifizierung von antiretroviralen Pharmaka aus Plasma, Serum, Vollblut und Dried Blood Spots (DBS) mittels LC/MS/MS Therese Koal 1, Heike Burhenne 1, Klaus Resch 1, Michal Svoboda 2, Volkhard Kaever 1 1

MH Hannover, Institut für Pharmakologie, Carl-Neuberg-Str. 1, D-30625 Hannover 2 Applied Biosystems, Frankfurter Str. 129 B, D-64293 Darmstadt

Schätzungsweise über 60 Millionen Menschen weltweit sind mit dem AIDS-Virus infiziert – davon erhalten weniger als 3% - hauptsächlich aufgrund der damit verbundenen, hohen Kosten – eine medikamentöse Therapie. Diese Therapie basiert auf der Gabe von Protease Inhibitoren (PI), Nukleosidischen Reverse Transkriptase Inhibitoren (NRTI) sowie NichtNukleosidischen Reverse Transkriptase Inhibitoren (NNRTI); es wird in allen Fällen eine Kombination dieser Präparate [1] gegeben. Der therapeutische Bereich dieser Pharmaka ist sehr eng: bei zu geringen Konzentrationen kommt es bald zur Entwicklung resistenter HIV-Stämme; ist die Konzentration zu hoch, treten vermehrt Nebenwirkungen wie Störungen im Fettstoffwechsel, Übelkeit, Erbrechen sowie Leberschäden auf. Zudem sind die interindividuell wirksamen Konzentrationsbereiche unterschiedlich. Eine quantitative Bestimmung der Medikamentenspiegel (Therapeutisches Drug Monitoring, TDM) ist aus diesem Grund äußerst hilfreich für die effiziente Dosisfindung für die Patienten [2, 3]. Für alle 9 der in Europa zugelassenen antiretroviralen Pharmaka der Klassen PI und NNRTI wurde eine quantitative LC/MS/MS-Methode entwickelt, und zwar für 7 PI (Saquinavir, Ritonavir, Indinavir, Nelfinavir, Amprenavir, Lopinavir und Atazanavir) und 2 NNRTI (Efavirenz und Nevirapin). Im Rahmen dieser Untersuchungen konnte gezeigt werden, daß man vergleichbare quantitative Ergebnisse sowohl aus Serum als auch aus Plasma und sogar aus Vollblut (nach Proteinfällung) erhält. Erste Untersuchungen an auf Filterpapier getrockneten Blutproben (Dried Blood Spots) deuten darauf hin, daß man die Bestimmung auch aus dieser Art von Probe durchführen kann – dies würde das Risiko einer Infektion für das mit diesen Proben befasste Laborpersonal erheblich verringern und einen vereinfachten Probenversand per Post ermöglichen. Literatur [1] Deutsche Apotheker Zeitung, 38 (2003) 89. [2] S.J. Soldin, N.Y. Rakhmanina, H.M.L. Spiegel, J.L. Sever, Ther. Drug Monit. 26 (2004) 107. [3] O.P. Soldin, R.J. Elin, S.J. Soldin, Arch. Pathol. Lab. Med. 127 (2003) 102.

160

Poster P54 Characterization of Plasma Protein Pools from Rheumatoid Arthritis Patients Treated by Immune Adsorption Therapy – A Proteomic Approach Towards Understanding the Active Principle C. Koy 1), S. Drynda 2), B. Ringel 1), J. Kekow 2), H. Montgomery 3), K. Tanaka 4), and M. O. Glocker 1) 1) Proteome Center Rostock, University of Rostock, Rostock, Germany. 2) Clinic of Rheumatology, University of Magdeburg, 39245 Vogelsang, Germany 3) Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu, UK 4) Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu, Japan Rheumatoid arthritis (RA) is a complex autoimmune disease whith chronic joint inflammation and subsequent progressive destruction of the articular tissue [1]. Rapid progress in molecular biology and genetic research led to the development of so-called ”biologicals”, such as Etanercept which sustainably reduce the disease activity in 60-70% of the patients (responders). Clearly, there is a need for alternative therapies for those patients that do not respond to current medication (non-responders) [2]. One alternative treatment for RA patients is an immune adsorption therapy [3]. Here, plasma proteins are adsorbed on an affinity column onto which protein A has been immobilized. Protein A displays a strong affinity to immunoglobulins. During therapy, plasma is transported through the column and then returned to circulation. During this treatment, (pathological) antibodies and immune complexes are removed in part from the circulation which may lead lead to remissions of symptoms with clearly reduced or even vanishing disease activity. The therapeutical principle of the immunoadsorption technique however is unclear. We applied a proteomic approach to visualize and identify proteins in the eluates of the affinity columns. 2D and 1D SDS gels were prepared with the protein extracts from the eluates, and spots and bands of interest were excised and digested with trypsin. Tryptic peptide mixtures were analyzed both directly by mass spectrometric peptide mass fingerprinting, and also by off-line LC-MALDI MS. Here, mass spectrometric sequencing of peptides enabled the identification of the present proteins. Initial results show that besides the expected immunoglobulins also other proteins e.g. apolipoproteins were found in the eluates. The role of these newly identified proteins for the progress of this therapy as well as their influence on the inflammation process shall be further investigated. Our results demonstrate that global proteomic approaches for characterizing complex biological systems on the protein level are becoming increasingly important in clinical and molecular medicine, e.g. in elucidating so far unclear therapeutical procedures. Literature [1] Drynda, S., Ringel, B., Kekow, M., Kühne, C., Drynda, A., Glocker, M.O., Thiesen, H.-J., and Kekow, J. Path. Res. Pract. 2004, 200, 165-171. [2] Glocker, M.O., Guthke, R., Kekow, J., and Thiesen, H.-J. Med. Res. Rev. 2005, submitted. [3] Furst, D., Felson, D., Thoren, G., Gendreau, R.M. Therapeutic Apheresis 2000, 4, 363-373.

161

Poster P55 Phosphohydroxyprolin ist eine proteinogene Aminosäure Axel Kühlberg, Mark Haid und Sabine Metzger Biologisch-Medizinisches-Forschungszentrum (BMFZ), Heinrich-Heine-Universität, Universitätsklinikum Düsseldorf, 40225 Düsseldorf [email protected] www.bmfz.de

Posttranslationale Modifikationen sind oftmals von entscheidender Bedeutung für die Struktur und Funktion von Proteinen. Anders als die Aminosäuresequenz der Proteine lassen sich die posttranslationalen Modifikationen nicht aus der genetischen Sequenz ableiten. Die Identifizierung der posttranslationalen Modifikation ist deshalb nur am Protein selbst möglich. Eine häufige Variante der posttranslationalen Modifikation ist die Phosphorylierung von Proteinen an den Seitenketten bestimmter Aminosäuren. Konservativen Schätzungen zufolge werden mehr als ein Drittel aller Proteine reversibel phosphoryliert [1]. Neben der O-Phosphorylierung der im Genom codierten Hydroxyaminosäuren Serin, Threonin und Tyrosin sind O-Phosphorylierungen an zwei weiteren Hydroxyaminosäuren möglich, die ihrerseits erst durch posttranslationale Modifikation, durch Hydroxylierung, entstehen: Hydroxylysin und Hydroxyprolin. Phosphoserin, Phosphothreonin und Phosphotyrosin wurden bereits vielfach in Proteinen identifiziert und auch ihre Position in der Aminosäuresequenz beschrieben [2, 3, 4]. Wenig untersucht ist bisher die O-Phosphorylierung von Hydroxylysin und Hydroxyprolin in Proteinen [5]. Die Identifizierung einer Sequenzposition fehlte bisher völlig. Mittels ESIMS/MS mit einem ESI-QqTOF gelang uns jetzt erstmalig der Nachweis von Phosphohydroxyprolin in dem Protein Alpha-Kristallin A aus der Augenlinse.

Literatur [1] [2] [3] [4] [5]

Herzig, S.; Neumann, J., Physiol Rev, 2000, 80: 173-210. McLachlin, D. T.; Chait, B. T., Curr Opin Chem Biol, 2001, 5: 591-602. Sickmann, A.; Meyer, H. E., Proteomics, 2001, 1: 200-206. Mann, M.; Jensen, O. N., Nat Biotechnol, 2003, 21: 255-261. Urushizaki, Y.; Seifter, S., Proc Natl Acad Sci U S A, 1985, 82: 3091-3095.

162

Poster P56 Development of a high-throughput procedure for the identification of protein-RNA cross-links from complex ribonucleoprotein particles (RNPs) based on IMAC and MALDI mass spectrometry 1

Eva Kühn1, Andrea Schmidt2, Reinhard Lührmann1, Henning Urlaub1 Max-Planck-Institut für Biophysikalische Chemie, Am Faßberg 11, 37077 Göttingen. 2 Gyros DE GmbH, Terminalstraße Mitte 18, 85356 München.

Ribonucleoprotein particles (RNPs) play essential roles in a number of fundamental cellular processes such as pre-mRNA splicing, rRNA maturation, post-transcriptional control (mRNA stability), RNA export, translation and translational control. Deciphering the molecular interactions within these particles is a prerequisite for the understanding of their function. In the absence of highly resolved 3D structures, direct UV cross-linking is the method of choice to gain insight into the molecular contacts between proteins and RNA. In recent years we have developed an approach that allows the "de novo" characterization of direct protein–RNA contacts sited in native and reconstituted RNPs [1]. Protein–RNA contacts in RNPs are made permanent by direct, UV-induced cross-linking. Subsequently, the protein and the RNA moieties are digested with various endoproteinases and ribonucleases and, in a final purification step, the resulting short peptide–RNA oligonucleotides are isolated by RP-HPLC, followed by MALDI-ToF mass spectrometry (MS) analysis to identify both peptide and RNA moiety. However, this type of analysis requires relatively large amounts of starting material and extensive purification of the cross-linked peptide– RNA heteroconjugates from the excess of non-cross-linked species to assign unambiguously the cross-linked heteroconjugates in the mass spectrometer. By combining capillary liquid chromatography and immobilized metal affinity chromatography (IMAC) [2], we have now established a procedure that allows the enrichment of cross-linked peptide–RNA oligonucleotides from low-abundance RNPs. Additional treatment of the IMAC-enriched samples with alkaline phosphatase (CIP) leads to the hydrolysis of the 3’-phosphate group of the RNA moiety of the cross-linked species. The loss of 80 Da in comparison with the untreated sample, as monitored in a MALDI-ToF MS spectrum, provides confirmation that the enriched mass peak is the actual peptide–RNA oligonucleotide cross-link and can thus be chosen for PSD sequence analysis. This overall strategy was then applied to the Gyrolab MALDI IMAC1 microlaboratory CD (Gyros AB, Uppsala, Sweden). The CD allows for the direct comparison of IMAC-enriched samples with samples that are additionally incubated with CIP in a fully automated and integrated process [3]. Sample enrichment, desalting, elution, and crystallization are carried out on the same device on the nanoliter scale, leading to highly concentrated and enriched sample preparations that are well suitable for PSD sequence analysis. Thus, application of the Gyrolab CD provides a possibility for the fast and efficient identification of protein– RNA contact sites in native RNPs of low abundance. Literatur [1] H. Urlaub, K. Hartmuth, R. Lührmann (2002), Methods 26 (2), 170-181. [2] E. K. M. Ueda, P. W. Gout, L. Morganti (2003), J. Chromatogr. A 988 (1), 1–23. [3] D. Hirschberg, T. Jagerbrink, J. Samskog, M. Gustafsson,, M. Stahlberg, G. Alvelius, B. Husman, M. Carlquist, H. Jornvall, T. Bergman (2004), Anal. Chem. 76 (19), 5864-5871.

163

Poster P57 Kinetic Energy Release am Beispiel ungewöhnlicher Doppelprotonentransferreaktionen

Alexandra E. Kulle 1, Stefan Schweiger 2, Guntram Rauhut 2 und Jürgen Grotemeyer 1 1

Inst. f. Phys. Chem. der CAU zu Kiel, Olshausenstr. 40, 24098 Kiel, [email protected]. 2 Inst. f. Theor. Chem. der Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart.

Am Beispiel des Pyrazol-Pyrazol-Komplexes und des Pyrazol-Guanidin-Komplexes wurden zwei Doppelprotonentransferreaktionen mit Hilfe quantenchemischer Methoden auf MP2/[aug]-cc-p-VDZ- und CCSD(T)/[aug]-cc-p-VDZ-Niveau theoretisch untersucht. Dabei weisen die berechneten Reaktionsprofile deutliche Unterschiede auf. Während im Fall des konzertiert synchron verlaufenden Doppelprotonentransfers am Pyrazol-Pyrazol-Komplex das Reaktionsprofil die Form einer Gauss-Kurve besitzt, besitzt der Doppelprotonentransfer im Pyrazol-Guanidin-Komplex ein außergewöhnliches Reaktionsprofil. An Stelle eines scharfen, eindeutigen Übergangszustandes tritt ein Gebiet konstanter Energie, ein so genanntes Plateau auf. Dies führt zu einem strukturlosen Übergangszustand [1]. Die Reaktionen wurden mit Hilfe des Zerfalls von metastabil gebildeten Ionen und der dabei auftretenden Verteilung der kinetischen Energie auf die auftretenden Produkte experimentell untersucht. Hierzu wurden MIKE-Spektren [2] aufgenommen. Aus den auftretenden Peakprofilen lassen sich deutliche Rückschlüsse auf den Prozess des Durchlaufens des Übergangszustandes sowie dessen Struktur machen. Dabei wird an den Signalen für die Fragmentionen die Peakform und die Breite, die der freigesetzten Energie (KER, kinetic energy release) [3] entspricht, ermittelt. Die Peakform nimmt dabei je nach dem Energieumverteilungsprozess unterschiedliche Strukturen an.

Literatur [1] S. Schweiger und G. Rauhut, J.Phys.Chem. A, 107, 9668 (2003). [2] R.G.Cooks, J.H. Beynon, R.M. Caprioli, G.R. Lester, Metastabile Ions, Elsevier, Amsterdam, 1973. [3] J. Laskin und C. Lifshitz, J. Mass Spectrom, 36, 459 (2001).

164

Poster P58 Atmospheric Pressure Photoionization (APPI)-MS-MS coupled with membrane assisted extraction for determination of pharmaceutical and endocrine disrupting compounds H. Paschke1, F. Lange2, S. Schrader1, M. Möder 1, W. Lorenz2 1

UFZ Centre for Environmental Research Leipzig-Halle, Department of Analytical Chemistry, Permoserstraße 15, 04318 Leipzig, Germany 2 Martin-Luther-Universität Halle-Wittenberg, Institut für Analytik und Umweltchemie K.-Mothes-Str.2, 06120 Halle, Germany The extremely low environmental concentration of micropollutants with endocrine disrupting effects or other bioactivities requires high analytical efforts. Appropriate analyte enrichment and separation from a complex matrix are some of the constraining time- and labor-consuming steps in sample preparation. The method presented combines APPI-MS-MS analysis and membrane assisted liquidliquid extraction to a highly sensitive procedure for the detection of trace concentration (ngµg/L) of UV-photoionizable compounds. APPI enables the ionization of a broader range of polar and semi-polar micropollutants otherwise only detectable with ESI-MS or APCI-MS or derivatization/GC-MS [1,2]. In order to optimize ionization efficiency and detection limits besides several dopants the influence of separation and matrix composition were tested. Furthermore, different types of membrane materials were examined for pre-concentration of pharmaceutical substances and musk fragrances from wastewater and surface water. Microporous polypropylene membranes with toluene as extraction solution proved to be suited for the determination of carbamacepine, fenofibrate, phenazone and caffeine with limits of detection of about 100ng/L. Membranes on polyethylene basis filled with ethylacetate enable enrichment of musk compounds such as Galaxolide and Tonalid (LOD about 50 ng/L). Mean concentrations of these pollutants occur in the range between 50 and 400 ng/L so a reliable monitoring is possible. Literatur [1] C.Zwiener, F.H. Frimmel, LC-MS analysis in the aquatic environment and in water treatment – a critical review, Part I: Instrumentation and general aspects of analysis and detection, Anal. Bioanal. Chem., (2004) 378: 851-861 [2] K. Reddersen, Th. Heberer, Multi-compound methods for the detection of pharmaceutical residues in various waters applying solid phase extraction (SPE) and gas chromatography with mass spectrometric (GCMS) detection, J. Sep. Sci. 2003, 26, 1443–1450

The work was supported by DFG Deutsche Forschungsgemeinschaft.

165

Poster P59 Charakterisierung der Struktur von Molekülionen und Untersuchung von Ion–Molekül-Reaktionen durch Ionenmobilitätsspektrometrie R. Laudien1, T. Beitz1, B. Kallies2, H.-G. Löhmannsröben1 1

Universität Potsdam, Institut für Chemie, Physikalische Chemie, Karl-Liebknecht-Str. 2425, D-14476 Golm. 2 Konrad-Zuse-Zentrum, Takustr. 7, D-14195 Berlin-Dahlem

Ionenmobilitäts (IM)-Spektrometrie ist eine Methode zur Untersuchung von Ion-MolekülWechselwirkungen und beruht auf der Driftbewegung von Molekülionen in einem Badgas unter dem Einfluß eines äußeren elektrischen Feldes. Die Mobilität in der Gasphase hängt wesentlich von der Geometrie der Ionen, die durch den Diffusionsquerschnitt der kollidierenden Partner beschrieben wird, und vom Wechselwirkungspotential zwischen den Badgasmolekülen und Ionen ab. IM-Spektrometrie ist somit eine Möglichkeit der experimentellen Bestimmung der Struktur von Molekülionen in der Gasphase. Dies wird an zwei Beispielen demonstriert. Im ersten Beispiel werden die Diederwinkel von aromatischen Molekülen, wie 9.10-Diphenylanthracen, 1.2.3- und 1.3.5-Triphenylbenzol, durch den Vergleich von berechneten und experimentellen Diffusionsquerschnitten abgeschätzt. Im zweiten Beispiel erfolgt eine Diskussion der Strukturen der Dimerkationen von ausgewählten PAK (Coronen, Perylen und Chrysen) im Hinblick auf eine Unterscheidung zwischen der Sandwich- und der T-Struktur der Dimere. Die gewonnenen Erkenntnisse werden auf höhere molekulare Oligomere der PAK-Kationen erweitert. Sowohl für die Dimere als auch die Oligomere wird der Abstand zwischen den Monomereinheiten abgeschätzt. In der IM-Spektrometrie bei Atmosphärendruck und Raumtemperatur liegt das Zeitinterval zwischen zwei Kollisionen im ps-Bereich. Damit ist die Vorraussetzung für effiziente IonMolekül-Reaktionen gegeben. Es werden erste Ergebnisse zu Protonentransferreaktionen von aromatischen Kationen (REMPI), wie Toluol+, auf neutrale Moleküle, wie Hexanon, präsentiert. Die Effizienzen der Protonentransferreaktionen von verschiedenen Alkylbenzolen und Alkylphenylethern werden verglichen.

166

Poster P60 Methionine Alkylation by Iodoacetamide May Interfere with Analysis of Peptide Serine/Threonine Phosphorylation by MS/MS Ralf Krüger, Chien-Wen Hung, Marina Edelson-Averbukh, Wolf D. Lehmann Central Spectroscopy, German Cancer Research Center, 69120 Heidelberg, Germany Proteins may show a molecular weight deviation of - 48 Da in ESI-MS [1] if alkylation of cysteine residues by iodoacetamide has been performed. This mass difference has been explained by an increase of +57 Da as result of alkylation at methionine [2] followed by neutral loss of 105 Da (2-(methylthio)acetamide) in the interface region. We have reinvestigated this phenomenon in view of its interference with spotting of pSer/pThr phosphopeptides by MS/MS based on their typical loss of H3PO4 (= 98 Da). The difference of about 8 Da between the loss of 105 Da from alkylated methionine versus the loss of 98 Da from pSer/pThr residues appears to allow for their clear discrimination. However, for multiply charged peptide ions, the difference is reduced to 8/z. For peptide ions with charge state 4+, this difference is reduced to 2. Typically, precursor ion selection in MS/MS experiments is performed with an m/z window of 2-3 Da. In case of an ambiguous precursor mass assignment, a loss of 105 from a peptide with charge state 4+ may be mistaken for the loss of 98 Da. Moreover, loss of 105 Da proceeds at low collision offset values, very similar to the characteristics of H3PO4 loss. In trace analysis of phosphopeptides from complex mixtures, the similarity of these two neutral loss processes increases the risk of false positive assignment of phosphorylation sites. In the Figure below, the MS/MS spectrum of the tryptic T22 ovalbumin peptide [ ILELPFASGT-Mox-S-Macetamido-LVLLPDEVSGLEQLESIINFEK + Na ]4+ is given which contains one oxidized and one alkylated Met residue. As demonstrated, the abundant loss of 105/4 (= 26.25) from the alkylated methionine residue has an appearance similar to the loss of 98/4 (= 24.5). [M+4H]4+ autopep_1069b 296 (50.494)

964.5

100

TO F Parent ES+ 40

990.0

%

0

m /z 600

700

800

900

1000

1100

1200

1300

1400

The +80 Da molecular weight shift of phosphorylation may also be mimicked by alkylation of methionine by iodoacetamide through its combination with sodium adduction (+ 57 Da + 22 Da = + 79 Da, as shown above). Since numerous studies on protein phosphorylation employ both Cys alkylation by iodoacetamide and neutral loss analysis, careful evaluation of MS and MS/MS data in phosphorylation analysis is recommended. References [1] Lapko VN, Smith DL, Smith JB. J. Mass Spectrom. 2000, 35, 572-575. [2] Gurd RN, Methods Enzymology 1972, 25, 424-438.

167

Poster P61 Multiplex Assay Monitoring by Means of ESI-MS for Proteolytic Activty Screening in Snake Venom André Liesener and Uwe Karst University of Twente, Chemical Analysis Group and MESA+ Institute for Nanotechnology, P.O. Box 217, 7500 AE Enschede, The Netherlands Employing ESI-MS as means of detection in enzymatic bioassays offers several advantages compared to the commonly used optical methods. The major difference is the opportunity to monitor by means of MS the conversion of several substrates by one enzyme in a single experiment. The concept of monitoring of enzymatic conversions by MS detection methods was introduced by Henion et al in 1989.[1] Since this first publication, the interest in this technique has been growing steadily. The majority of papers published deals with investigations on one-enzyme-single-substrate assays. The first approach towards a multiple-substrate assay was presented by Gerber et al. in 1999.[2] In a recent publication, Leary et al. showed the first competitive MS-based multiple-substrate assay, in which the conversion of four substrates by one enzyme was investigated.[3] In this paper, the development of a MS-based multiplex assay scheme for the monitoring of proteolytic reactions is described. The experimental set-up comprises of a flow injection system coupled to an ion trap MS for the detection. As model system, five different proteases involved in the blood coagulation cascade were selected. The simultaneous conversion of five different substrates by each of the enzymes was monitored. The resulting activity profiles were mapped and used as reference. As real sample, 19 snake venom fractions were tested for their enzymatic activities. The activity maps were obtained for all of these and the proteolytic activity of each fraction towards the different substrates was evaluated. The semi-quantitative assessment of the activity maps allowed a ranking into five different classes (very high, high, medium, low and no activity). Literatur [1] E.D. Lee, W. Mueck, J. Henion, T.R. Covey, J. Am. Chem. Soc. 1989, 111, 4600–4604. [2] S.A. Gerber, C.R. Scott, F. Turecek, M.H. Gelb, J. Am. Chem. Soc. 1999, 121, 1102–1103. [3] N. Pi, J.A. Leary, J. Am. Soc. Mass Spectrom. 2004, 15, 233–243.

168

Poster P62 Proteomics on Zebrafish Embryos Vinzenz Link, Carl-Philipp Heisenberg, Andrej Shevchenko Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany Gastrulation defines the first process in the development of all multicellular organisms where an uniform mass of cells transforms into different tissues (germ layers) with specific properties. Developmental biologists have so far mainly focused on the genetic network that triggers this process. In contrast, our approach aims at identifying the key proteins that define the specific characteristics of the different germ layers. The protein expression patterns of the ectodermal and mesendodermal germ layers of the model organism zebrafish (D. rerio) were compared by 2D-gel electrophoresis in combination with the DIGE system. Since the zebrafish protein database is still incomplete a layered strategy is applied for the identification of spots: Initially all spots are subjected to MALDI peptide mass fingerprint (PMF) analysis. Low scoring hits are confirmed by LC-MS on an ion-trap. Spots that can not be identified with these methods due to the incomplete database coverage are subjected to ESI-quadrupole-TOF analysis for the generation of partial sequences. The MS-BLAST [1] search algorithm is applied to identify these spots based on homology. This approach exploits the speed of PMF search and complements it, when necessary, with the power of homology based mass spectrometry. Literature [1] Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time-of-flight mass spectrometry and BLAST homology searching. Shevchenko A, Sunyaev S, Loboda A, Shevchenko A, Bork P, Ens W and Standing KG Anal Chem. 2001;73(9):1917-26

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Poster P63 Analysis of membrane proteins in mouse brain – The power of multi-dimensional separation Christiane Lohaus, Andreas Nolte, Helmut E. Meyer, Katrin Marcus Medical Proteom-Center, Ruhr-University of Bochum, Germany Due to their crucial role in many cellular processes, their direct association with human disorders, and beyond this their prevalent extracellular accessibility for drugs the understanding of the function of membrane proteins is of fundamental importance. However, their analysis presents a special challenge largely due to their highly hydrophobic nature. They are not readily soluble in polar solvents and often undergo aggregation. In 2DPAGE based proteomic studies membrane proteins are underrepresented due to their insufficient transfer from the first to the second dimension. In the present study within the framework of the Human Brain Proteome Project (HBPP) new technologies most efficient for functional proteome analysis - particularly membrane proteins - were established as alternatives to 2D-PAGE. The ability to characterize a complex protein sample by mass spectrometry (MS) depends on the power and sensitivity of the separation techniques employed prior to the MS analysis. The high complexity and the large dynamic range involved in analyzing protein mixtures, make a multi-dimensional separation necessary. A 3D-system for protein/peptide separation was chosen after fractionated sample preparation. The membrane protein fraction was first separated by 1DPAGE. The protein bands were cut out from the gel and tryptically digested. After extraction the peptides were further separated by strong cation exchange chromatography (SCX). Resulting fractions were off-line transferred to a nano-RP(reversed phase) HPLC and are online analyzed by ESI-Iontrap-MS/MS. 473 non-homologous proteins including 55 with at least one transmembrane helix (11.6 %) and 74 membrane associated proteins (15.6 %) - in total 129 (27.3 %) proteins - could be identified with this 3D-LC-MS/MS approach. Comparing these experiments with a 2DPAGE-MS/MS approach it results in a lower number of identified proteins but a higher quantity of transmembrane proteins. 3D-LC-MS/MS proves to be complementary to 2DPAGE-MS/MS and even better for the analysis of membrane proteins but is limited by the complexity of the sample. Due to this, the duty cycle of the mass spectrometer enables only the detection and fragmentation of a small peptide fraction. Thus, 3D-LC-MS/MS  proves to be complementary to 2D-PAGE-MS/MS  is not qualified for „Shotgun“ proteome analysis of high complex peptide mixtures  is not a solution for low abundant proteins  on protein basis is a very useful tool with the possibility of combining it with quantitative methods.

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Poster P64 Flugzeiteffekte in der topologischen Massenspektrometrie Kai Maaß 1, Werner Bouschen 1, Arne Leisner 1, Bernhard Spengler1 1

Institut für Anorganische und Analytische Chemie, Justus-Liebig-Universität Gießen, Schubertstr. 60 Haus 16, 35392 Gießen.

Ziel ortsaufgelöster mikroskopischer Oberflächenanalytik ist die mehrdimensionale Darstellung der chemischen Zusammensetzung einer Probe. Eine gezielte Vorverarbeitung und statistische Auswertung der gemessenen massenspektrometrischen Daten erlaubt eine schnelle und präzise Beschreibung der Topologie [1] von biologischen oder technischen Proben mit einer Auflösung im Submikrometerbereich. Insbesondere zur exakten Bestimmung der z-Koordinate sind umfangreiche Vergleichsmessungen und Kalibrierungen notwendig. Die Messungen wurden mit dem am Institut entwickelten hochauflösenden 3D-SMALDIMassenspektrometer LAMMA 2000 [2] durchgeführt. Gegenüber der publizierten Konfiguration wurde zur Verbesserung der Ortsauflösung ein frequenzverdreifachter Nd:YLF-Laser mit einer Wellenlänge von 349 nm verwendet. Die deutlich höhere Repetitionsrate (50 – 100 Hz) ermöglicht zudem kürzere Messzeiten. Für die Probenpositionierung wurde ein 3D-Piezotisch in das Gerät integriert, der eine laterale Auflösung von 250 nm in x/y-Richtung bzw. 50 nm in z-Richtung besitzt. Mit dieser Messanordnung kann die Topologie in einem Flächenbereich von 100 x 100 µm erfasst werden. Zur Kalibrierung des Höhenprofils kann der Piezotisch in z-Richtung maximal 7 µm verfahren werden. Die Positionierung der Probe entlang der Beschleunigungsachse z kann zusätzlich zur Messung von Flugzeiteffekten genutzt werden. Exponierte Regionen nichtleitender Proben zeigen dabei eine Flugzeitverlängerung durch eine geringere Energieaufnahme bis zum Verlassen der Beschleunigungsstrecke. Die gleichzeitig verkürzte Flugstrecke spielt beim gewählten Aufbau des Massenspektrometers nur eine untergeordnete Rolle für die Gesamtflugzeit. Die 3D-Erweiterung der SMALDI-MS gestattet es, Moleküle mit Massen größer 1000 u nicht nur lateral, sondern auch topologisch mit Auflösungen im Submikrometerbereich zu vermessen. Mit Hilfe des nachgeschalteten, automatisierten Datenverarbeitungssystems können die bis zu 320000 Spektren eines Scans für beliebig viele ausgewählte Massensignale zu Verteilungsbildern umgesetzt werden. Vergleiche mehrerer Laserpulse auf dieselbe Probenposition liefern dabei Informationen über den Materialabtrag während der Messung. Das Projekt wurde gefördert vom Bundesministerium für Bildung und Forschung, Aktenzeichen 0312834A. Literatur [1] Patentanmeldung: B. Spengler, W. Bouschen, K.-P. Hinz, „Dreidimensionale Abbildung der chemischen Oberflächenzusammensetzung von Objekten“, Aktenzeichen: DE 103 10 518.23. [2] B. Spengler, M. Hubert: “Scanning microprobe matrix-assisted laser desorption ionization (SMALDI) mass spectrometry: instrumentation for sub-micrometer resolved LDI and MALDI surface analysis.”, J. Am. Soc. Mass Spectrom. 13 2002 735-748. [3] K. Maaß, W. Bouschen, B. Spengler: “Development of Topological Mass Spectrometry in Surface Microanalysis”, Desorption 2004, St. Petersburg, Russland, 31: August – 3. September 2004.

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Poster P65 Structural characterization of Shiga toxin 1-binding glycosphingolipids in crude TLC overlay assay-derived silica gel extracts by nanoESI-QTOF mass spectrometry Iris Meisen1,2, Alexander W. Friedrich3, Helge Karch3, Jasna Peter-Katalinić1, and Johannes Müthing1 1

Institute for Medical Physics and Biophysics, University of Münster, Robert-KochStrasse 31, D-48149 Münster. 2 Institute of Occupational Medicine, University of Münster, Robert-Koch-Strasse 51, D-48149 Münster. 3 Institute for Hygiene, University of Münster, Robert-Koch-Strasse 41, D-48149 Münster

Shiga toxin (Stx)-producing Escherichia coli have been identified as a worldwide cause of serious human gastrointestinal disease and the life-threatening hemolytic uremic syndrome [1]. In this study a combined strategy of preparative thin-layer chromatography (TLC) overlay assay and mass spectrometry was established for the detection and structural characterization of Stx1-binding glycosphingolipids (GSLs). Neutral GSLs from human erythrocytes were separated on silica gel precoated TLC plates and probed for the presence of Stx1-receptors. Stx1-positive bands were extracted with chloroform/methanol/water (30/60/8, v/v/v) as reported previously for antibody-stained GSLs [2]. These crude extracts were used without any further purification for the structural characterization of Stx1-binding GSLs by nanoESI-QTOF mass spectrometry in the negative ion mode. In all extracts investigated, neutral GSLs were detected as singly charged deprotonated molecular ions [M – H]-, and neither buffer-derived salt adducts nor coextracted contaminants from the overlay assay procedure or the silica gel layer were observed. For the structural characterization of Stx1-receptors low-energy CID mass spectrometry was applied to high and even low abundant species. All MS/MS spectra obtained, contained full series of Y-type ions, B-type ions and additional ions generated by ring cleavages of the sugar moiety. Only analytical quantities in the microgram scale of a complex GSL mixture were required for the full structural mass spectrometric characterization of Stx-1-ligands as Gb3Cer (CD77) and Gb4Cer. Parallel experiments using anti-Gb3Cer and anti-Gb4Cer specific antibodies in TLC overlay assays confirmed these results. This effective TLC-MS-joined procedure offers a wide scope of clinical applications for any carbohydrate binding agents such as bacterial toxins, plant-derived lectins [3] or human proteins with yet unknown or broad binding specificity [4]. Literature [1] Karch,H. (2001) Semin. Thromb. Hemost. 27, 207-213. [2] Meisen,I., Peter-Katalinić,J., and Müthing,J. (2004) Anal. Chem. 76, 2248-2255. [3] Müthing,J., Meisen,I., Kniep,B., Haier,J., Senninger,N., Neumann,U., Langer,M., Witthohn,K., Milosević,J., and Peter-Katalinić,J. (2005) FASEB J. 19, 103-105. [4] Schlossmacher,M.G., Cullen,V., and Müthing,J. (2005) New Engl. J. Med., in press.

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Poster P66 Mesoporöse TiO2-Membranen als MALDI-Probenträger zur Analyse von Phosphopeptiden in Peptidgemischen Anna Melikyan1, Thomas Moritz2, Michael W. Linscheid1 1

Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor Str. 2, 12489 Berlin; 2 Department of Applied Analytical Development, Analytics WW Autotech Germany.

Die mesoporösen Titanoxid-Filme[1] können zur selektiven Immobilisierung und Anreicherung[2] von Phosphopeptiden aus Peptidgemischen benutzt werden. Bringt man solche Titanoxid Filme auf leitfähige Substrate auf, können sie als Affinitätsprobenträger für die MALDI Massenspektrometrie dienen. Im Gegensatz zu IMAC-säulen (Immobilized Metal Affinity Chromatography) ist mit den entwickelten Affinitätsprobenträgern eine „on target“- Probenvorbereitung möglich. Die Probenträger können für die Bioanalytik im High Troughput Screening auf dem Gebiet der Phospho-Proteomics genutzt werden. Die Experimente wurden mit den Phosphoproteinen α-Casein, β-Casein und Bovine Fetuin durchgeführt. Nach Anreicherungsexperimenten mit den tryptischen Verdauen konnten Phosphopeptide nachgewiesen werden. Als leitfähiges Substrat wurde einseitiges ITObeschichtetes (Indium Tin Oxide) Glass benutzt. Um eine reproduzierbare Methode für die Anwendung der Affinitätsprobenträger für die Phospho-Proteomics zu entwickeln, wurden weitere leitfähige Substrate im Betracht gezogen. Es wurden Probenträger für MALDI Massenspektrometrie aus Aluminium und Silizium angefertigt, die über eine Spot-Größe von 0,5 bzw. 1 mm verfügen. Um die Oberfläche des mesoporösen Titanoxid Films zu benutzen, wurden in das Trägermaterial erst Vertiefungen geätzt und dann der Film aufgebracht. Dadurch erhält man eine bessere Wechselwirkung zwischen dem Film und der Probe, aus der die Phosphoproteine selektiv angereichert werden können. Ergebnisse der Messungen bzw. Spektren werden in dem Beitrag vorgestellt Literatur: [1] Xu, Qunyin; Anderson, M. A.; J. Mater. Res., 1991, 6, 1073-1081 [2] Moritz, Thomas; Doktorarbeit, 2003, Institut für Chemie Humboldt Universität zu Berlin.

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Poster P67 Vergleich verschiedener Enzyme auf Eignung für die massenspektrometrische Analyse von Membranproteinen Björn Meyer 1, Sandra Hofmann 1, Carsten Corvey 1, Khalid Siddiqui 2, Bernd Ludwig 2, Michael Karas 1 1

Institut für Pharmazeutische Chemie, J. W. Goethe-Universität, Marie-Curie-Str. 9, 60439 Frankfurt am Main, 2 Institut für Biochemie, J. W. Goethe-Universität, Marie-Curie-Str. 9, 60439 Frankfurt am Main Der Analyse von Membranproteinen kommt eine besondere Bedeutung zu, da 20-30% des Genoms von jedem Organismus Membranproteine codieren. Die Identifizierung dieser wichtigen Proteine ist mit den etablierten massenspektrometrischen Methoden meistens möglich, da ein aussagekräftiges MS/MS-Spektrum normalerweise für die Wiederfindung in Datenbanken ausreicht. Für die Charakterisierung von posttranslationalen Modifikationen oder Mutationen ist aber eine höhere Sequenzabdeckung nötig. Es konnte beispielsweise gezeigt werden, dass die Kombination von Trypsin und CNBr [1] oder der Einsatz von Proteinase K [2] zu einer signifikanten Verbesserung der Sequenzabdeckung führt. Für diese Studie wurden als Modellsysteme das Membranprotein Cytochrom c1 (Thermus thermophilus) und der Membranproteinkomplex Cytochrom c oxidase (Paracoccus denitrificans) verwendet. Cytochrom c1 hat nur eine transmembrane α-Helix (1 TMH). Cytochrom c oxidase ist aus vier Untereinheiten aufgebaut, die 12 TMH (ca. 62 kDa), 2 TMH (33,5 kDa), 7 TMH (30,6 kDa) und 1 TMH (5,4 kDa) enthalten. Beide Modellsysteme wurden in E. coli expremiert, daraus isoliert, wieder mit Detergentien gelöst und enzymatisch verdaut. Unterschiedliche Proteasen (Trypsin, Chymotrypsin, Proteinase K, Elastase) werden hierfür unter verschiedenen Bedingungen (pH, Zeit, Puffer) gegenüber gestellt. Die resultierenden Peptide werden direkt mit MALDI-TOF- und MALDITOF/TOF-MS sowie nach chromatograpischer Trennung auf einem nano-HPLC-System mit ESI-MS und ESI-MS/MS analysiert. Vergleichskriterien der Enzyme untereinander sind Art und Umfang der Sequenzabdeckung sowie die Sequenzinformation aus den MS/MSSpektren als auch die Eignung der Daten für eine Datenbanksuche.

[1] Brat A. van Montfort, Mark K. Doeven, Benito Canas, et al., Combined in-gel tryptic digestion and CNBr cleavage for the generation of peptide maps of an integral membrane protein with MALDI-TOF mass spectrometry, Biochimica et Biophysica Acta, 1555: 111-115 (2002) [2] Christine C. Wu, Michael J. MacCoss, Kathryn E. Howell, John R. Yates III, A method for comprehensive proteomic analysis of membrane proteins, Nature Biotechnology, 21: 532-538 (2003)

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Poster P68 SELDI-profiling and Identification of Differently Expressed Proteins in Body Fluids from Children Suffering from Juvenile Idiopathic Arthritis M. Meyer1, R. Bogumil1, K. Falk2, B. Ringel2, I. Prüfer2, R. Nowack3, J. Oppermann3, and H.-J. Thiesen4 1

Ciphergen Biosystems GmbH, Göttingen, Germany Proteome Center Rostock, Medical Faculty, University of Rostock, Rostock, Germany 3 Carl-Thiem-Klinikum Cottbus, Cottbus, Germany 4 Institute of Immunology, Medical Faculty, University of Rostock, Rostock, Germany

2

Juvenile idiopathic arthritis (JIA) is an inflammatory disease of childhood with highest prevalency among chronic inflammatory diseases in children. We investigated protein profiles of plasma and of synovial fluid (SF) from eight patients with JIA in a differential manner. Body fluids were first subjected to weak cationic exchange ProteinChip ® Arrays (CM10) without purification. Protein expression difference analyis of mass spectra showed distinct and reproducible differences. According to SELDI-analysis, the abundance of an ion signal at 6.6 kDa was found higher in plasma than in SF (p < 0.00001). Further differences in abundances between the body fluids were observed with ion signals at 51 kDa (p < 0.01), at 66.6 kDa (p < 0.0001), and at 100 kDa (p < 0.001). These latter protein ion signals were more intense in SF than in plasma. In a second step, samples were pooled and subjected to fractionation using a SELDI assisted protein purification strategy in an attempt to identify the differently expressed proteins. With the help of strong anionic exchange column materials (BioSepra® Q HyperD® F), the samples could be fractionated such that the 6.6 kDa protein was found predominantly in the flow-through and in the organic fraction (co-eluent isopropanol, acetonitrile, TFA). The flow-through was further fractionated on reversed-phase beads (BioSepra® Poly BeadsTM) and the fractions eluted with 30% and 40 % acetonitrile in 0.1% TFA were enriched in the 6.6 kDa marker and subjected to SDS-PAGE resulting in approx. 12 bands in each lane after Coomassie staining. One half of the band at approx. 7 kDa was eluted as intact protein out of the gel to verify the molecular weight and the second half was forwarded to tryptic in-gel digestion and mass spetrometric peptide mass fingerprinting (PMF). PMF together with MS/MS analysis of three selected ion signals clearly resulted in the identification of the 6.6 kDa protein as apolipoprotein C1. Similarly, the samples from both body fluids were fractionated such that the 51 kDa protein eluted in the pH4 fraction from strong anion exchange column material. The fractions were further analyzed by SDS-PAGE and protein bands with molecular masses in the 50 kDa range were subjected to PMF and to MS/MS analysis of selected ion signals after tryptic digestion, respectively. In one band from SF, Zinc-alpha-2-glycoprotein precursor (P25311) was identified by sequencing two peptide ion signals. In a protein band from plasma, alpha-2-HS-glycoprotein precursor (Fetuin-A; P02765) and in another band, again from SF, Vitamin D-binding protein precursor (P02774) and Ig gamma-1 chain C region – human (gi|70028) were identified, each with one sequenced peptide ion signal. The Vitamin D-binding precursor has a molecular weight of 51.2 kD (without its signal chain) and fits therefore well with the expected mass of the 51 kD signal. The 66.6 kDa signal was not analyzed further, but most likely refers to human serum albumin. The roles of the respective proteins in SF need to be further investigated.

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Poster P69 Mass spectrometric analyses of cyanobacterial proteomes: Studies of the salt stress response and the phosphoproteome of Synechocystis PCC 6803 Stefan Mikkat1, Sabine Fulda2 and Martin Hagemann3 1

Universität Rostock, Medizinische Fakultät, Core Facility Proteomanalytik, JoachimJungius-Str. 9, 18059 Rostock, Germany, 2,3 Universität Rostock, FB Biowissenschaften, Pflanzengenetik2 und Pflanzenphysiologie3, Albert Einsteinstr. 3a, 18051 Rostock, Germany The cyanobacterium Synechocystis sp. strain PCC 6803 has become a widely used model organism for studies on cellular physiology and molecular biology, especially in relation to salt and osmotic stress response [1]. We have investigated salt stress-induced changes in protein synthesis pattern after salt shock (short-term response) and the changed protein composition in salt-acclimated cells (long-term response) of the soluble protein fraction. Autoradiography of 2-DE separations of 35S-methionine labeled proteins was used to visualize the proteins that were induced during the short-term response. The long-term response was investigated by differential analysis of Coomassie-stained 2-DE gels from salt-adapted versus control cells. After image analysis, differentially expressed proteins were excised from the gels, digested with trypsin and identified by peptide mass fingerprinting with MALDI-TOF MS. Most of the proteins induced in salt-shocked cells (short-term response) are involved in the general stress response (e.g. different chaperones, superoxide dismutase, Clp protease). The increased synthesis of these proteins correlates well to data from transcriptom analyses [2,3]. After long-term salt acclimation, numerous proteins were accumulated compared to control cells. Some of them are salt specific (e. g. proteins involved in osmolyte synthesis) but also changes in many enzymes belonging to basic metabolic pathways were detected. These changes are only partly reflected by transcriptom analysis. Protein phosphorylation plays an important role in signal transduction and enzyme regulation. It has been shown that protein phosphorylation pattern changed in salt-treated cells of Synechocystis [4]. We have started an analysis of the “phosphoproteom” of Synechocystis using the new fluorescent Pro-Q Diamond phosphoprotein gel stain. Until now, approx. 50 putative phosphoproteins were identified by peptide mass fingerprinting of excised and digested spots. Many of the newly identified phosphoproteins act in basic metabolic pathways, but their phosphorylation needs confirmation. However, one of the most intensely stained protein spots after phosphoprotein staining was identified as phosphorylated form of the nitrogen regulatory protein P-II (GlnB), a well characterized phosphoprotein [5]. The nonphosphorylated form of GlnB was also identified in the same gel after general protein staining. The comparison of the MALDI spectra from both spots showed the phosphorylation-caused mass increase of 80 Da for a tryptic peptide that covers the known phosphorylation site at Ser-49 residue of the Synechocystis GlnB protein. [1] Burja AM, Dhamwichukorn S, Wright PC (2003) Trends in Biotechnol 21: 504-510 [2] Kanesaki Y, Suzuki I, Allakhverdiev SI et al. (2002) Biochem Biophys Res Commun 290: 339-348 [3] Marin K, Kanesaki Y, Los DA et al. (2004) Plant Physiol 136: 3290-3300 [4] Hagemann M, Golldack D, Erdmann N, Biggins J (1993) FEMS Microbiol Lett 113: 205-210 [5] Forchhammer K, Tandeau de Marsac N (1995) J. Bacteriol 177 : 5812-5817

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Poster P70 Identification of N-glycosylation sites and characterisation of N-glycan structure of soluble intercellular cell adhesion protein (Lec1 sICAM-1) by MALDI-FTICR-MS A. Moise1, E. Damoc1, V. Otto2, T. Schürpf2, G. Folkers2 and M. Przybylski1 1

Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz 2 Department of Chemistry and Applied Biosciences Swiss Federal Institute of Technology Zurich Cell adhesion molecules in the immune system play an important role in lymphocyte-target cell conjugate formation. One such molecule, intercellular adhesion molecule 1 (ICAM-1), is important in the function, aggregation, and adherence of leukocytes. Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein. ICAM-1 is also released as a soluble glycoprotein (sICAM-1), consisting of the extracellular part of membrane-bound ICAM-1. Some studies suggest that sICAM-1 is a competitive inhibitor of leukocyte adhesion to vascular endothelium, whereas others suggest that sICAM-1 has signalling functions. It was shown recently that the signalling function of sICAM-1 in astrocyte strongly depends on its glycosylation. The recent development of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry enabled a breakthrough for high resolution mass spectrometric structure analysis. FT-ICR mass spectrometry has the unique sensitivity and high mass accuracy necessary for the characterisation of post-translational modifications. In order to identify the glycosylation sites of sICAM-1, the protein expressed in Lec1 cells, was purified by affinity chromatography, resolved by SDS-PAGE, subjected to PNGase-F / Endo-H in-gel deglycosylation, trypsin digestion, and direct analysis by MALDI-FTICR-MS. To determine the possible N-glycan composition corresponding to the experimentally determined masses of trypsin generated glycopeptides from sICAM, the GlycoMod programme was used. MALDI-FTICR-MS provided the unequivocal identification of seven glycosylation sites, with mass determination accuracies lower than 8 ppm. The N-glycan structure found in Lec-1 sICAM-1 consists of two GlcNAc and five mannose residues. References: [1] Damoc E, Youhnovski N, Crettaz D, Tissot JD, Przybylski M. (2003) Proteomics Aug;3(8):1425-33 [2] Otto V, Schürpf T, Folkers G, Cummings R. D, (2004) J. Biol. Chem. Aug 20;279(34):35201-9 [3] Albach C, Damoc E, Denzinger T, Schachner M, Przybylski M, Schmitz B. (2004) Anal Bioanal Chem. Feb;378(4):1129-35.

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Poster P71 Infrared Multiphoton Dissociation of peptides released by in-capillary proteolysis M. Mormann, G. Pohlentz, S. Kölbl, and J. Peter-Katalinić University of Münster, Institute for Medical Physics and Biophysics, Münster, Germany Tryptic peptides obtained from in-gel or in-solution digests are commonly used for the identification by mapping or sequencing of proteins. We have recently described a new strategy that allows the direct analysis of tryptic peptides by nanoelectrospray Q-TOF mass spectrometry during the proteolytic digest in the electrospray capillary [1]. In the present study this approach will be extended to the detection of the tryptic peptides by use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Small- to medium size proteins, e.g., apomyoglobin or ribonuclease B, were dissolved in 10 mM ammonium hydrogen carbonate containing 10% methanol. Subsequently, trypsin was added, the mixture was transferred to an electrospray capillary and mass spectra were acquired directly. The high mass accuracy and unique resolving power achieved by the FT-ICR mass analyzer allow simultaneous detection and identification of numerous peptide ions released upon proteolysis according to their accurate mass. Typically, mass accuracies of 1-2 ppm were achieved when internal calibration was applied. The sequence coverage obtained from this experiments was usually 90% or higher giving rise to a high probability score if no modifications are present in the analyte species. For more detailed analysis of the species under inspection, sequence tags of the tryptic fragments were generated by infrared multiphoton dissociation (IRMPD). Selected peptide precursor ions formed by in-capillary proteolysis of apomyoglobin were isolated in the ICR cell and submitted to IRMPD. As expected, IRMPD gave mainly rise to b- and y-type ions accompanied by the neutral loss of either water or ammonia as secondary fragmentation processes. An additional significant application of this approach is glycoproteomics. When low abundant N-glycosylated peptide ions from ribonuclease B were excited by IR photons the cleavage of glycosydic bonds gave rise to the most abundant and diagnostic fragment ions.

In conclusion, the analysis of peptides released upon in-capillary proteolysis by use of an FT-ICR mass spectrometer in combination with IRMPD tandem MS experiments represents a powerful tool for the identification of proteins and their modifications. References [1] G. Pohlentz, S. Kölbl, J. Peter-Katalinić, Proteomics, in press

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Poster P72 Untersuchungen am Ablationsprozess der MALDI durch Postionisation Tassilo Muskat1, Dirk Walbrodt2 und Jürgen Grotemeyer3 Inst. f. Phys. Chem. der Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, 24098 Kiel, 1 [email protected] 2 [email protected] 3 [email protected] Die MALDI gehört inzwischen zu den Routine-Analytikverfahren. Dies ist insoweit überraschend, da die grundlegenden Prozesse bisher weitestgehend unverstanden sind. Bisher wurde für die Matrixauswahl im wesentlichen auf empirische Daten zurückgegriffen. Vor allem die Ionisations- bzw. Kristallisationseigenschaften einer Matrix wurden als Auswahlkriterien herangezogen. Die Ablationseigenschaften wurden weitestgehend unberücksichtigt gelassen. Wie aber die gemachten Untersuchungen zeigen, sind diese bei den einzelnen Matrizes recht unterschiedlich. So ergeben sich auch Auswirkungen für zusammen mit der Matrix untersuchte Analyte. Um die Ablationseigenschaften der Matrizes zu untersuchen, wurden die Geschwindigkeiten der ablatierten Neutralteilchen untersucht. Hierzu wurde zeitversetzt zum Ablationslaser eine Nachanregung (Postionisation) durch einen zweiten Laser innerhalb der Ablationswolke (Plume) hervorgerufen. Die Ablation ist jetähnlich und lässt sich am ehesten mit einer verschobenen Maxwell-BoltzmannGeschwindigkeitsverteilung beschreiben. Als charakteristische Werte können z.B. die wahrscheinlichsten Geschwindigkeiten ermittelt werden. Weiterhin kann aus der Breite der Verteilung die Temperatur des Plumes ermittelt werden. Durch Postionisation ablatierter, neutraler Analytmoleküle kann ebenso die Auswirkungen der Expansion auf diese untersucht werden. Tatsächlich wird eine Kühlung der Analytmoleküle vorgefunden. Untersucht man nun die Plumeentwicklung durch Variation von Laserleistung, Innendruck der Quellregion oder des Postionisationsortes, so wird eine ausgedehnte Entwicklung noch bis zu 1.4mm nach dem Ablationsort festgestellt. In der Literatur wurde dagegen bisher von einer kompakten Plumeentwicklung ausgegangen. Durch Stoßprozesse, die während dieser langen Plumeentwicklung stattfinden, ist aber auch eine ausgedehnte Ionenbildung möglich. Diese später erzeugten Ionen tragen zum Untergrundrauschen bei. In der Literatur wurden Matrixmoleküle und -cluster als Quelle des sogenannten chemical noise identifiziert. Die Entstehung dieser Verunreinigung der Spektren lässt sich mit den gefundenen Ergebnissen ebenso erklären, wie der Erfolg der Atmosphären-Druck-MALDI. Bei dieser wird durch den erhöhten Hintergrunddruck ein kompakter Plume ausgebildet und die lang andauernden Stoßprozesse vermieden.

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Poster P73 Higher sample information content by combing magnetic bead based fractionation and LC-MALDI-TOF/TOF on complex biological samples Dagmar Niemeyer, Sören-Oliver Deininger, Arndt Asperger and Marcus Macht Bruker Daltonik GmbH, Permoserstr. 15, 04318 Leipzig, Germany MALDI-TOF mass spectrometric analysis of peptides and proteins is highly dependent on sample composition and purity. Especially the wide variety of biological samples such as blood serum/plasma, tissue lysates or cell culture supernatants used in the field of clinical proteomics often requires additional sample preparation steps prior to MS analysis due to their high complexity. Isolation, enrichment and purification of peptides and proteins from complex biological material can be achieved by magnetic bead based chromatography dedicated to various functional groups [1]. The goal of this straight-forward approach which is fully integrated in Bruker Daltonics clinprot automation platform is to analyse large sample sets resulting in reproducible peptide profiles. A software supported comparison [2] of these datasets can result in condition-specific fingerprints that may significantly support disease diagnoses as well as evaluation of disease stages, progression or treatment efficacy. A new strategy for increasing peptide profile complexity per sample on the one hand and for supporting the immediate identification of interesting peptides by MALDI-TOF/TOF on the other hand is to combine the magnetic bead based sample pretreatment with a capillary LC run. For this human serum was prepared with magnetic beads of different functionalities. One part of the eluate recovered from the magnetic beads was used directly for automatic MALDI-TOF (ultraflex) acquisition of profile spectra and the other part was applied on a capillary LC system with RP column. Fractions recovered from RP column were spotted on a MALDI-TOF target and equally analyzed automatically by ultraflex MALDI-TOF MS. Acquired mass spectra were evaluated by a sophisticated software package for generation of a compound list containing all peptides assigned to fraction number, position on target and a compound score reflecting the respective peak intensity. Based on this compound score peptides were selected for an automated MALDI-TOF/TOF analysis. A combination of all MS spectra of all LC-fractions resulted in a profile spectrum with increased complexity. The peak number in the mass range of up to 4 kDa could be multiplied approximately by a factor of 4. Additionally several peptides could be identified easily by MALDI-TOF/TOF analysis and a subsequent MASCOT database search including immediate identification of posttranslational modifications such as phosphorylations.

References [1] J. Villanueva, J. Philip, D. Entenberg, C.A. Chaparro, M.K. Tanwar, E.C. Holland, P. Tempst, Serum peptide profiling by magnetic particle-assisted, automated sample processing and MALDI-TOS mass spectrometry, Anal. Chem. 76 (2004) 1560-1570. [2] Ralf Ketterlinus, Sen-Yung Hsieh, Shih-Hua Teng, Helen Lee, and Wolfgang Pusch, Fishing for biomarkers: analyzing mass spectrometry data with the new ClinProTools software, BioTechniques, in press

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Poster P74 Einsatz von LC/ESI-MS und GC/MS zur Bestimmung ausgewählter Pharmaka und ihrer Metaboliten J. Nolte1, W. Nigge1, M. Bataineh1/2, M. Grote1 1

ISAS-Institute For Analytical Sciences; Postfach 101352, 44013 Dortmund. 2 Universität Paderborn, Department Chemie, Warburger Str.100, 33098 Paderborn

Unerwünschte Immissionen von Pharmaka sowie deren Metaboliten aus den Anwendungsbereichen der Human- und Veterinärmedizin ins Ökosystem sind von zunehmender Bedeutung für die Wasserqualität. Steigende Konzentrationen der weltweit angewandten Wirkstoffe Sulfamethoxazol, Carbamazepin, Diclofenac und Ibuprofen sowie deren Metaboliten in vielen Oberflächengewässern, in einigen Grundwässern und sogar schon vereinzelt im Trinkwasser sind ein eindeutiger Hinweis darauf, dass derartige Wirkstoffe in den Klärprozessen nur anteilig erfasst werden [1-3]. Als Konsequenz daraus ist es neben einer Verbesserung der Klärtechniken umso dringender, zuverlässige analytische Techniken zur Verfügung zu haben. Eine Kombination aus Chromatographie und Massenspektrometrie erfüllt die Erwartungen am besten. Deshalb wurde die Leistungsfähigkeit der Mikro-LC/ESI-MS, GC/MS und MS/MS Technik am Verhalten der oben genannten Analyte in Abhängigkeit unterschiedlicher Testbedingungen untersucht. Die Untersuchungen mit LC/MS wurden mit einem Ion Trap Massenspektrometer vom Typ Finnigan LCQ Deca, der HPLC Einheit Spectrasystem P400 und der analytischen Kapillarsäule Aquasil C18 durchgeführt. Für die Analyte Carbamazepin, Diclofenac und Ibuprofen konnte die Empfindlichkeit um einen Faktor 10 gesteigert werden, indem eine kleine Anreicherungskartusche (5 mm lang, 300 µm Innendurchmesser, gefüllt mit C18Material) in-line vor die analytische Säule installiert wurde. Die Pharmaka und deren Metaboliten lassen sich in einem Analysenlauf trennen. Es muss aber berücksichtigt werden, dass der pH-Wert der Probe einen immensen Einfluss sowohl auf die Retentionszeit als auch auf die Trenneigenschaften hat. Einzelheiten zum Analysenverfahren werden berichtet und diskutiert. Zur Absicherung der Ergebnisse wurde auch die MS/MS Technik angewandt. Parallel zu den Arbeiten mit LC/MS wurde auch GC/MS, Typ HP GCD G 1800 A, eingesetzt. Als analytische Säule diente eine HP-5MS Kapillarsäule. Da einige der Analyten relativ polar sind, ist ein Derivatisierungsschritt empfehlenswert. Die Ergebnisse zu verschiedenen Derivatisierungsreagenzien bezüglich ihrer generellen Eignung für alle Wirkstoffe werden präsentiert ebenso wie die Bestimmungsgrenzen.

Literatur [1] D.G. Kennedy, R.J. McCracken; J Chromatogr A, 812 (1998) 327-337 [2] M. Meisenheimer, T.A. Ternes; Vom Wasser, 94 (2000) 203-212 [3] J.A. Jönsson, L. Mathiasson; J. Sep. Sci., 24 (2001) 495-507

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Poster P75 Epitope Elucidation of C-terminal Amyloid Precursor Protein (C-APP) Specific Antibodies of Alzheimer's Disease by High Resolution Mass Spectrometry Gabriela Paraschiv, Xiaodan Tian, and Michael Przybylski Department of Chemistry, Analytical Chemistry, University of Konstanz, 78457 Konstanz, Germany;

The proteolytic degradation of the transmembrane type I protein, amyloid precursor protein (APP) to neurotoxic ß-amyloid polypeptide fragments, accumulating in extracellular plaques, is one of the characteristics of Alzheimer’s disease (AD). Right after the membrane region of APP follows the APP cytoplasmic or intracellular domain, which consists of 47 aa. Important features of APP intracellular domain include its interaction with different neuronal proteins and phosphorylation at several residues, which have been shown to increase Aß generation by cleavage of APP. Therefore, knowledge of the cytoplasmic domain of APP is important for understanding the formation of Aß at the molecular level In this study, intracellular APP polypeptide was synthesized by solid phase peptide synthesis using Fmoc chemistry, which then was used for epitope elucidation using two specific antibodies; one monoclonal anti-Cter antibody (Jonas; JmAb) and a polyclonal antiCter APP antibody. Epitope-excision/extraction mass spectrometry was used for the epitope mapping by combination of specific proteolytic digestion and peptide mapping with FTICRMS (epitope-excision and –extraction) [1]. The epitopes were determined to be located at the C-terminal APP (740-747) for the monoclonal antibody and C-terminal APP (727-737) for the polyclonal antibody, and the epitopes were confirmed by ELISA binding assays and authentic synthetic peptides [2]. The epitope structures are efficient tools for studies of intracellular degradation pathways of APP and C-APP, and might be used for development of new specific vaccines of AD. References: 1. 2.

Suckau, D., Mak, M. and Przybylski, M. Proc Natl. Acad. Sci. USA 89, 5630 (1992). Tian, X., Cecal, R., McLaurin, J., Manea M., Stefanescu, R., Grau, S., Harnasch, M., Amir, S., Ehrmann, M., St. George-Hyslop, P., Kohlmann, M. and Przybylski M. Eur. J. Mass Spectrom. in press (2005).

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Poster P76 Monitoring nucleation reactions by coupling different reactors to an ESI Mass Spectrometer: about silicate species and zeolithe precursors Pelster S., Schüth F., Schrader W. Max-Planck Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr Condensation of monomers leads to oligomeric units that conglomerate further more in different nucleation steps to bigger species. When the crystallization point is reached the growth of the crysal can be observed macroscopically. The knowledge of these processes involved in the formation of a solid is rare and limited and often restricts to empirical findings. Better understanding of solid state formation could help to design more efficient heterogeneous catalysts like zeolithes. But while it is possible to analyze the precursors and even the solids using a number of different analytical methods, the observation of the nucleation process is still problematic. We have developed a procedure to analyze inorganic aqueous silicates with different mass spectrometers utilizing electrospray-ionization [1, 2]. Although the working conditions are beyond standard conditions due to high salt content of the samples in the percent range, it was possible to characterize a number of different species and to address questions such as charge and degree of hydrolysis of the species present in solution. To be able to get further insight into the silicate chemistry and especially the nucleation process we have coupled a reactor to a mass spectrometer. This combination allows a detailed and time-resolved study of the behavior of silicates in solution with regard to their environment. To study different reaction conditions we used four silicate reactants such as alkoxysilanes and three counter-ions in regard to the formation of different silicate oligomers. The nature of the alkoxysilanes affects basically the reaction rate caused by slower or faster hydrolysis. Thus, choosing the adequate alkoxy lenght enables detailed observation of every interesting reaction step in the nuceation process. The counter-ions are composed from tetraalkylammoniumhydroxyd which plays a major role in the formation of the silicate species. Adding specific crosslinkers, thermal treatment and adjusting the pHvalue leads to conditions closer to the crystallization point, where larger species are formed that are supposed to be precursors for the zeolithe synthesis [3, 4]. Literatur [1] Bussian P., Sobott F., Brutschy B., Schrader W., Schüth F., Angew. Chemie Int. Ed., (2000) 39 (21), 39013905; Angew. Chemie, (2000) 112 (21), 4065 [2] Pelster S., Schrader W., Schüth F., in preparation [3] Agren P., Thomson S., Ilhan Y., Zibrowius B., Schmidt W., Schüth F., Stud.Surf.Sci.Catal., (2002) 142, 159-166 [4] Ravishankar R., Kirschhock C.E.A., Schoeman B.J., Devos D., Grobet P.J., Jacobs P.A., Martens J.A., Proceedings of the 12th Zeolithe Conference MRS, Pennsylvania, (1999), 1825-1832

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Poster P77 Bestimmung der Elementarzusammensetzung von Fullerenderivaten mittels hochauflösender API-oTOF-Technologie Matthias Pelzing1, Nadine Bucci2, Bianca Flock2, Jürgen H. Gross2, Thomas J. J. Müller2 1

2

Bruker Daltonik GmbH, Permoserstr. 15, D-04318 Leipzig Organisch-Chemisches Institut der Universität, Im Neuenheimer Feld 270, D-69120 Heidelberg

Phenothiazine sind elektronenreiche Heterotricyclen, die als reversible Redoxsysteme fungieren können.1,2 Durch Anknüpfung von [60]Fulleren konnten wir PhenothiazinFulleren Diaden erhalten,3 die sich als interessante Kandidaten für molekulare Schalter ansehen lassen, die auf photoinduziertem Elektronentransfer (PET) beruhen. Bisher wurde hauptsächlich Matrix-Assisted Laser Desorption/Ionization zur Charakterisierung eingesetzt und – sofern es die Löslichkeit gestattete – Fast Atom Bombardment zur HR-MS. Vier dieser [60]Fullerenderivate wurden nun zur Erweiterung des methodischen Repertoires vor allem mit Hinblick auf eine Vereinfachung der HR-MS mit Electrospray Ionisation (ESI), Atmospheric Pressure Photoionisation (APPI) und Atmospheric Pressure Chemical Ionisation (APCI) an einem orthogonalen TOF-Massenspektrometer untersucht. Dabei lieferte jede dieser Ionisierungsmethoden im positiv-Modus Quasimolekülionen durch Protonierung. Die APPI-Spektren waren im Vergleich zu den ESI-Spektren weniger komplex und wiesen ein deutlich besseres Signal- zu Rauschverhältnis für das Quasimolekülion auf. Als nachweisstärkste Ionisierungsmethode für die vorliegenden Proben erwies sich APCI. Vor- und Nachteile der verschiedenen Ionisierungstechniken werden diskutiert. Durch den Einsatz neuer Analog zu Digital-Wandler (ADC) und optimierter Ionenoptik werden mit dem verwendeten Bruker micrOTOFfocus™ die Spektren generell hochaufgelöst (RFWHM > 10000) und bei entsprechender Kalibrierung massengenau (Fehler < 5 ppm) erzeugt. Darüber hinaus enthalten sie auch die präzisen Isotopenverhältnisse (± 2 % bezogen auf intensivsten Isotope) und die akkurate Masse für alle Isotopomere. Aus den gemessenen akkuraten Massen aller Isotopomere und deren Intensitätsverhältnissen wird mittels entsprechender Software (Generate FormulaTM) eine statistische Abweichung (Sigma ValueTM) des gemessenen Isotopenmusters vom theoretischen Wert bestimmt. Diese zusätzliche Information stellt eine weitere Dimension für die Bestimmung der Elementarzusammensetzung dar und erlaubt, die Liste der möglichen Elementarzusammensetzungen für eine experimentelle akkurate Masse deutlich zu reduzieren. Im konkreten Fall der Fullerenderivate ließen sich in Abhängigkeit von der Molmasse aus 800–1700 möglichen Summenformeln im Massenfenster von ± 5 ppm unter Berücksichtigung des Sigma-Wertes die Verbindungen eindeutig zuordnen bzw. die Liste auf 2-3 Kandidaten einschränken. Diese Technik ermöglichte außerdem eine einfache Identifizierung von wenig intensiven Signalen durch Solvensaddukte und Oxidationsprodukte. Literatur 1 M. Sainsbury in Comprehensive Heterocyclic Chemistry (Eds. A. R. Katritzky, C. W. Rees) Pergamon Press, Oxford, 1984, Vol. 3, S. 995. 2 McIntyre, H. Gerischer, Ber. Bunsen Ges. Phys. Chem. 1984, 88, 963. 3 M. Maggini, G. Scorrano, M. Prato, J. Am. Chem. Soc. 1993, 115, 9798.

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Poster P78 Analysis of synthetic chelating agents in surface and waste water by ion chromatography mass spectrometry Thomas P. Knepper 1) , Andreas Werner 1) and German Bogenschütz 2) 1) Europa Fachhochschule Fresenius, Limburger Strasse 2, D-65510 Idstein 2) Deutsche Metrom AG, In den Birken 3, 70794 Filderstadt, Germany

Coupling of ion chromatography with electrospray mass spectrometry (IC-MS) is a simple, sensitive and quick method for the determination of polar organic traces in water samples without derivatization. Analysis of the chelating agents ethylenediamino tetraacetate (EDTA) and diethylenetriamino pentaacetate (DTPA) in aqueous samples was done by ICMS on an anion exchange column after simple sample preparation steps. Quantification down to a concentration level of 1µg L-1 even in wastewater influents and effluents was achieved utilizing 13C marked internal standards and measuring the individual [M-H+]- and stable [M-4H+ + Fe3+]- cluster ions. The method was validated against certified, but more time consuming routine methods. Applying this method a series of several European water samples were analyzed upon EDTA and DTPA indicating their nature as polar persistent pollutants.

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Poster P79 Vergleich zweier massenspektrometrischer Detektoren hinsichtlich ihrer Empfindlichkeit gegenüber Pestiziden und die Untersuchung von Fragmentierungsmustern Manuela Peschka, Thomas P. Knepper, Europa Fachhochschule Fresenius, Limburger Strasse 2, D-65510 Idstein Es wurde eine quantitative Analysenmethode für eine Reihe ausgewählter Pestizide, chlorierte Kohlenwasserstoffe, Phosphorsäureester und Triazine, entwickelt. Mit Hilfe dieser Methode konnten zwei massenspektrometrische Detektoren (MSD) hinsichtlich ihrer Empfindlichkeit miteinander verglichen werden. Bei einem der beiden MSD handelt es sich um ein Gerät, dessen Ionenquelle aus einem inerten Material besteht. Dieses Material soll verhindern, dass aktive Substanzen, wie Drogen oder Pestizide, von der Quelle adsorbiert werden, was zu einem Verlust an Empfindlichkeit führt. Für welche Art Analyten eine Empfindlichkeitssteigerung zu verzeichnen ist, wird anhand der durchgeführten Untersuchung aufgezeigt. Der Massenanalysator besteht hier aus einem Quadrupol. Bei dem Massenanalysator des zweiten MSD handelt es sich um eine Ionenfalle, welche aus üblich verwendetem Edelstahl besteht. Um die beiden MSD miteinander vergleichen zu können, wurden über 100 Kalibriergeraden erstellt, anhand deren Steigungen die Empfindlichkeit gegenüber den einzelnen Substanzen ermittelt wurde. Die höchste Empfindlichkeitsdifferenz zu Gunsten des MSD mit der inerten Ionenquelle ergab sich für niedermolekulare Dithiophosphate, wie Disulfoton oder Demeton-S. Weiterhin wurden einige Massenspektren der ausgewählten Pestizide miteinander verglichen, um eventuelle Tendenzen im Fragmentierungsverhalten festzustellen. Hierbei konzentrierte man sich hauptsächlich auf die Gruppe der Dithiophosphate. Es wir aufgezeigt, dass funktionelle Gruppen die Fragmentierung der Verbindung beeinflussen. Die daraus erhaltenen Informationen können bei der Strukturaufklärung unbekannter Verbindungen oder bei der Suche nach Metaboliten hilfreich sein.

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Poster P80 Untersuchungen zum Auftreten von Barbituraten in deutschen Oberflächengewässern Manuela Peschka, Jan Eubeler, Thomas P. Knepper, Europa University of Applied Sciences Fresenius, 65510 Idstein, Germany; [email protected] Um Einträge in die Mulde und deren weiteren Verbleib in der Umwelt zu untersuchen wurde eine empfindliche Gaschromatographie-Massenspektrometrie (GC-MS) - Screening Methode entwickelt. Dabei konnten erstmals in einem Oberflächengewässer verschiedene Barbiturate in höheren Konzentrationen halbquantitativ nachgewiesen werden. Bei den detektierten Verbindungen handelt es sich um sedative Hypnotika mit einer beruhigenden Wirkung auf das Zentralnervensystem (ZNS), im Einzelnen um Butalbital, Pentobarbital, Secobarbital und n-Allylbarbital (siehe Abbildung 1). Da diese Barbiturate wiederholt in mehreren Proben vorkamen, wurde eine quantitative Analysenmethode für diese Stoffe etabliert. Bei zwei Vertretern der Verbindungsklasse wurden mehrfach Konzentrationen im µg/L detektiert. Aufgrund der betäubenden Wirkung der Barbiturate und der gefundenen Konzentrationen (> 1µg/L) von Butalbital und Pentobarbital wurde die Mulde intensiver untersucht, mit dem Ziel, mögliche Eintragpfade zu und weitere aktive Metabolite zu identifizieren. Desweiteren wurden Abbauversuche zu diesen neu in der aquatischen Umwelt detektierten Verbindungen durchgeführt.

O

NH

O

O

NH

O

O

O

O

O

1

O NH

NH

NH O

NH

2

N

NH O

3

Abbildung 1: Strukturformeln der Barbiturate Butalbital (1), Pentobarbital (2), Secobarbital (3), n-Allylbarbital (4)

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O

4

Poster P81 Analyse eines wässrigen Extraktes von Früchten der Gattung Aronia mittels HPLC-MS und FTICR-MS Stefan Pieper 1, Michael W. Linscheid 1 1)

Humboldt-Universität zu Berlin, Institut für Chemie, Brook-Taylor-Str.2, 12489 Berlin

Der für dieses Projekt zur Verfügung gestellte Fruchtextrakt entstammt den Früchten der Gattung Aronia (Apfelbeere). Diese Gattung stammt ursprünglich aus dem Osten Nordamerikas und ist mittlerweile auch in Gebieten Mittel- und Nordeuropas heimisch geworden. Bei den Früchten handelt es sich um rundliche violettschwarze Beeren, die sich aus den weißen Blüten bilden. Allgemein werden die Aroniabeeren für ihren hohen Gehalt und ihr breites Spektrum an Vitaminen und Mineralien sehr geschätzt. Speziell den polyphenolischen Bestandteilen in diesen Beeren wird ein hohes Potential als Antioxidanz zugesprochen [1]. Deshalb finden sie Einsatz als Inhaltsstoffe für medizinische Vitaminpräparate oder auch für Medikamente gegen Kinderkrankheiten, Leber- und Gallenerkankungen. Bei der zu analysierende Probe handelt es sich um einen dickflüssigen wässrigen dunkelviolettfarbenen Extrakt, welcher mittels massenspektrometrischer Methoden mit Schwerpunkt auf HPLC-IT-MS und FTICR-MS, untersucht wurde. Hierbei wurde besonderes Augenmerk darauf gelegt, die polyphenolischen Bestandteile des Extraktes zu analysieren und mittels hochauflösender Massenspektrometrie deren Zusammensetzung zu bestimmen. Neben den polyphenolischen Bestandteilen wurden auch verschiedene andere Verbindungen detektiert, z.B. Polyhydroxycarbonsäuren. [1] –

C. Manach, A. Scalbert, C. Morand, C. Remesy, L. Jimenez, Am. J. Clin. Nutr., 2004, 79, 727-747

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Poster P82 LC/APCI-MS in Lipid Analysis Klaus Raith, Hany Farwanah, Christian Brenner, Christian Schmelzer, and Reinhard Neubert Martin-Luther-Universität Halle-Wittenberg, Fachbereich Pharmazie, Institut f. Pharm. Technologie u. Biopharmazie, W.-Langenbeck-Str. 4, 06120 Halle (S.), Germany. Atmospheric Pressure Chemical Ionisation (APCI) is a well-established ionisation technique for liquid samples. Although LC/MS is mostly carried out using the electrospray interface, APCI and the relatively new Atmospheric Pressure Photoionisation are viable alternatives. ESI and APCI are complementary in lipid analysis. Whereas polar lipids such as phospholipids or many sphingolipids are preferably ionised by electrospray, APCI comes into play for less polar compounds. We have investigated the oxidation of cholesterol during food processing. A new reversed phase LC/APCI-MS method was developed for 25-hydroxycholesterol, cholestane-3β-56β-triol, 7β-hydroxycholesterol, 7-ketocholesterol, 5,6-epoxycholesterol, and cholesterol. In another study the ceramides of human stratum corneum were analysed using normal phase LC/APCI-MS. In this case, the use of APCI instead of ESI was mostly motivated by the compatibility to normal phase solvents with low polarity. Furthermore, the most lipophilic ceramide species such as ceramide [EOS] show a considerably increased response with APCI. In conjunction with an ion trap mass analyzer, structural elucidation was possible using MS/MS. In general, APCI is a versatile, reliable ionisation method in lipid analysis. Care should be taken to prevent artefact formation due to the loss of water or carbon dioxide, if applicable. Whereas older APCI interfaces show optimal performance at high flow rates around 1 ml/min, new interfaces can operate with lower flow rates as obtained using LC columns with 1 or 2 mm ID. References [1] K. Raith, C. Brenner, H. Farwanah, G. Müller, K. Eder, and Reinhard H.H. Neubert: A new LC/APCI-MS method for the analysis of cholesterol oxidation products in food. J. Chromatogr. A, in press. [2] H. Farwanah, P. Nuhn, R. Neubert, and K. Raith: Normal Phase LC Separation of Stratum Corneum Ceramides with Detection by Evaporative Light Scattering and APCI Mass Spectrometry. Anal. Chim. Acta 492 (2003), 233-239. [3] K. Raith, H. Farwanah, and R. Neubert: Lipidomics of Human Stratum Corneum Ceramides: Online LC/APCI-MS versus Offline HPTLC/ESI-MS/MS. Proceedings of the 52nd ASMS Conference, Nashville, TN, USA, 2004. [4] K. Raith and R. Neubert: Structural Studies on Ceramides by Electrospray Tandem Mass Spectrometry. Rapid Commun. Mass Spectrom. 12 (14/1998), 935-938. [5] H. Farwanah, K. Raith, R. Neubert and J. Wohlrab: Ceramide profiles of the uninvolved skin in atopic dermatitis and psoriasis are comparable to those of healthy skin. Arch. Dermatol. Res., submitted.

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Poster P83 MATI-Spektroskopie an Pyrimidin Mikko Riese1 und Jürgen Grotemeyer2 1

Inst. f. Phys. Chem. der CAU zu Kiel, Olshausenstr. 40, 24098 Kiel, [email protected] 2 Inst. f. Phys. Chem. der CAU zu Kiel, Olshausenstr. 40, 24098 Kiel, [email protected] Mit Hilfe der MATI-Spektroskopie lassen sich die Zustände von Ionen exakt bestimmen. Sie beruht auf dem Prinzip der ZEKE-Spektroskopie mit dem Unterschied, dass hier anstelle der Energie der Photoelektronen die Intensität des Ionensignals bestimmt wird. Die Ionisation erfolgt mittels REMPI über resonante Zwischenschritte. Die elektrisch angeregten Singulett und Triplett Zustände von Pyrimidin waren Gegenstand zahlreicher experimenteller [1,2] und theoretischer [3,4] Untersuchungen. Bei diesen Untersuchungen gibt es jeweils nur eine Arbeit die den ionischen Grundzustand D0 mittels PIE-Spektroskopie [5] und ZEKE-Spektroskopie [6] betrachtet. Daher wurden MATI-Spektren aufgenommen, um mehr über den ionischen Grundzustand D0 zu erfahren. Als Zwischenzustände wurden zusätzlich zu denen, die bei der ZEKESpektroskopie verwendet wurden, weitere gewählt, um eine vollständigere Untersuchung des ionischen Grundzustandes zu erhalten. Des weiteren wurden quantenchemische Rechnungen sowohl für den Grundzustand S0 den ersten elektrisch angeregten Zustand S1 wie den ionischen Grundzustand D0 durchgeführt. Im Rahmen des Posters werden die Spektren diskutiert, die erhaltenen Banden den molekularen Bewegungen zugeordnet und mit den ZEKE-Spektren verglichen.

[1] Fischer, G.; Cai, Zheng-Li; Reimers, J.R., Wormell, P.; J.Phys.Chem. A, 2003, 107, 3093 [2] Knight, A. E. W.; Jones, J. T. ;Parmenter, C.S. ; J. Phys. Chem. 1983, 87, 973 [3] Öhrn, A.; Christiansen, O.; Phys. Chem. Chem. Phys. 2001, 3, 730 [4] Buma, W. J., Donckers, m. C. J. M., Groenen, E. J. J., J. Am. Chem. Soc. 1992, 114, 9544 [5] Mikami, N. Sugahara, Y., Ito, M.m J. Phys. Chem. 1986, 90, 2080 [6] Sato, S., Omiya, K., Kimura, K. J. Elect. Spec. rel. Phen. 1998, 97, 121

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Poster P84 Mass Spectrometric Identification of Low Abundant Proteins from Human Plasma and Synovial Fluid after Depletion of Albumin by Free-Flow-Electrophoresis B. Ringel 1, S. Drynda 2, S. Mikkat 1, C. Koy 1, I. Schinke 3, G.J. Eppert 3, H.-J. Thiesen1, J. Kekow 2 and M.O. Glocker 1 1

Proteome Center Rostock, University of Rostock, Joachim-Jungius-Str. 9, 18059 Rostock, Germany. 2 Clinic of Rheumatology, University of Magdeburg, 39245 Vogelsang, Germany. 3 SEPSERV GmbH, Helmholtz-Str. 29, 10587 Berlin, Germany. We investigated plasma and synovial fluids from patients with Rheumatoid Arthritis and controls in order to identify disease-related marker proteins. However, the separation and identification of low abundant proteins in body fluids is strongly hampered by the presence of so-called bulk proteins such as albumin, immunoglobulins, and transferrin which represent more than 80% of the total protein content in plasma. In order to overcome present limitations, we fractionated the plasma and synovial fluids protein mixtures by continuous preparative isoelectric focusing using a free-flow electrophoresis system [1-4]. With this method albumins were removed efficiently. Afterwards, albumin-reduced fractions were directly used or concentrated by ultra-filtration and precipitation, respectively. Free-flow fractions were separated by either 2D-electrophoresis or by HPLC followed by 1Delectrophoresis for further separating proteins out of these mixtures. The obtained protein bands / protein spots that are to some extent representing so-called low abundant proteins were visualized by Coomassie blue and silver staining, respectively. After excision and ingel digestion, the peptide mixtures were analyzed by MALDI ToF and MALDI QIT ToF mass spectrometry. Together with an improvement concerning the low protein abundance level that is now analytically reached with less interference of the main bulk proteins, we are now more and more confronted with contaminations that are inherently accompanied with gel electrophoresis and subsequent work-up procedures. In our experience faint protein spots in the gels consist of the protein of interest plus traces of spreading proteins of higher abundance. Another source of contaminants in faint proteins spots are organic polymers that are co-eluted together with the tryptic peptides and which yield in strong ion signals in the mass spectra, hence, also interfering with identification through peptide mass fingerprinting. Despite the mentioned obstacles and by interactively interpreting all mass spectra individually, we were able to identify a number of low abundant proteins and so-called ”tissue leakage” proteins such as calgranulins A and B, insulin-like growth factor binding protein, and serpin B3, respectively, for which the plasma concentrations are reported to be below 0.1 mg / L. Some of the newly identified proteins may be worthwile to be clinically evaluated for their potential to function as RA marker proteins in addition to those that have already been reported [5]. References [1] Free-flow Electrophoresis 1990 (K. Hannig, H. G. Heidrich, eds.), GIT Verlag, Darmstadt. [2] B. Ringel, M. O. Glocker, G. Weber, G. Kekow, and H.-J. Thiesen, in Conf. Proc. 4th. Siena Meet., 2000. [3] M. O. Glocker, C. Koy, P. Serrano-Fernandez, M. Kreutzer, S. Möller, B. Ringel, S. Mikkat, R. Martin, S. Drynda, J. Kekow, H.-J. Thiesen, in Conf. Proc. 6th Siena Meeting, 2004. [4] G. Weber, J. Bauer, Electrophoresis 1998, 19, 1104-1109. [5] Drynda, S., Ringel, B., Kekow, M., Kühne, C., Drynda, A., Glocker, M.O., Thiesen, H.-J., and Kekow, J. Path. Res. Pract. 2004, 200, 165-171.

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Poster P85 New tools for nanoLC-FTICR-MS measurements Andreas Römpp 1,2, Ioana Taban 2, Mark Duursma 2, Vinh An Thieu 1, Dieter Kirsch 1, Bernhard Spengler 1 and Ron Heeren 2 1 2

Institute of Analytical Chemistry, Justus Liebig University, D-35392 Giessen, Germany. FOM Institute for Atomic and Molecular Physics (AMOLF), Amsterdam, The Netherlands

The combination of nano-scale liquid chromatography with Fourier Transform Ion Cyclotron Mass Spectrometry (FTICR) offers unsurpassed quality (and quantity) of analytical data. We present strategies to use this promising technology in a flexible and efficient way. A number of functionalities can be made available through the internet. Collaborations within the scientific community can be facilitated by the use of remote experimentation, i.e. control of measurement systems through a network connection irrespectively of the geographic location. Thus expensive or unique instrumentation can be used without the need to travel. We also present analysis software that can be run in a distributed computing environment in order to speed up postprocessing of mass spectral data. This is particularly important for the large data sets generated by hyphenated FTICR measurements. Another methodology that can be applied to nanoLC-FTICR data is composition based sequencing (CBS). It allows the determination of the amino acid sequence of unknown peptides on the basis of MSn data without employing protein database information. The benefits of the described strategies are demonstrated by two biological applications. Cerebrospinal fluid from breast cancer patients was analyzed in order to find diagnostic biomarkers. Furthermore biomolecules were separated from the surface of human renal carcinoma cells in order to identify cancer specific MHC class I bound peptides for development of vaccines or other immune-based forms of treatment.

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Poster P86 Kopplung eines Ionenmobilitätsspektrometers (IMS) an ein QuadrupolMassenspektrometer (Q-MS) mittels eines Interface zur Reduktion von Atmosphärendruck zum Hochvakuum Karsten Rowold1, Mikko Riese2, Uwe Eggers, Michael Karstens und Jürgen Grotemeyer3 1

Inst. f. Phys. Chem. der CAU zu Kiel, Olshausenstr. 40, 24098 Kiel, [email protected] 2 [email protected] 3 [email protected]

Anwendungsorientierte Massenspektrometer (MS) sollen im Allgemeinen robust, klein und transportabel sein und eine hohe Auflösung besitzen. Ferner wächst das Interesse nicht nur an Masse/Ladungs-Verhältnissen messbar mittels MS, sondern auch an der GasphasenIonenmobilität, die mit dem Ionenmobilitätsspektrometer (IMS) ermittelt wird. Diese beiden Größen lassen sich zu einer zweidimensionalen Matrix zusammenfassen, die eine Analyse von Molekülen vereinfacht. Das Ziel der hier vorgestellten Arbeit war es, ein industriell erhältliches IMS weitestgehend unverändert mit einem bauformkleinen und ebenfalls standardmäßig erhältlichen Quadrupol-Massenspektrometer (Q-MS) zu koppeln. Hierzu trugen auch die Vorteile der verwendeten Tritium (3H)-Ionisationsquelle (e.g. geringe Baugröße, keine äußere elektrische Beschaltung sowie konstante Ionisation) bei. Das hierbei entwickelte Interface zwischen IMS und Q-MS besteht im Wesentlichen aus einem dreistufigen Pumpstufensystem aus Vor-, Zwischen- und Hochvakuum, wobei im Besonderen Wert auf hohe Ionentransmission sowie geringe Pumpleistung und daraus resultierende kleine Vakuumpumpen gelegt wurde. Die Entwicklung dieses IMS-Q-MS umspannt dabei das Verständnis der Abhängigkeit der Ionen-Trajektorien vom IMS-Potentialgefälle (homogen/inhomogen), der –Driftgasströmungsdynamik und dem damit verbundenen Auflösungsvermögen, aber auch den ebenfalls von diesen Eigenschaften abhängigen Transmissionsunterschieden hin zum Quadrupol. Weiterhin wurden verschiedene Pinholes (Ionenaustrittsöffnung aus dem IMS in die erste Vakuumstufe) und unterschiedliche elektrostatische Linsensysteme (zur Leitung und Fokussierung des Ionenstromes) untersucht, um ein besseres Verständnis über die Ionenausbeute nach durchlaufenen Vakuumstufen zu erhalten. Parallel zum experimentellen Fortschritt dieses Gerätes wurden simulierte Ergebnisse (mittels Simulationssoftware SimIon V7.0) stetig in die Entwicklung einbezogen. Entwicklungsschritte und Erkenntnisse sowie das Gesamtkonzept werden mit Eckdaten präsentiert und kritisch diskutiert.

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Poster P87 Thermostable trypsin derivates for enhanced in-gel digestion in high throughput proteomics Natalie Schmalz1, Marek Šebela2, Taťána Drhlíková2, Henrik Thomas1, Andrej Shevchenko1 1

2

MPI of Molecular Cell Biology and Genetics, Dresden, Germany Palacký University, Department of Biochemistry, Olomouc, Czech Republic

Identification of proteins after electrophoretic separations in gel is typically achieved by their enzymatic in-gel digestion and analyzing the recovered peptides by mass spectrometry. Bovine trypsin is an aggressive protease with high cleavage specificity, which is used in proteomics research for the digestion of proteins. However, known disadvantages of bovine trypsin are its weak thermostability and fast autolysis. Those problems could be overcome by chemical modification of lysyl groups in trypsin, for example using reductive methylation. We synthesized trypsin modified by di-, tri-, tetrasaccharides and cyclodextrins and demonstrated that their thermostability and autolysis resistance was much higher than those of native and methylated trypsins. To characterize their catalytic efficiency in in-gel digestion of proteins, we studied the kinetics of digestion, primary cleavage specificity and autolysis patterns in line with a “fast digestion method” previously established in our laboratory (Havlis et al, 2003). The kinetics was studied by MALDI TOF MS using O18-labeled internal standards for quantifying the yield of digestion products.

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Poster P88 MALDI Imaging of Proteins and their Interaction Partners on Surfaces Christian E.H. Schmelzer, Andreas Huenerbein, Klaus Raith und Reinhard H.H. Neubert Institute of Pharmaceutics and Biopharmaceutics, Martin Luther University HalleWittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale) [email protected] Quartz crystal microbalance (QCM) is a technique used to characterise specific interactions such as enzyme/substrate, antigen/antibody, or carbohydrate/lectin interactions by measuring changes in resonance frequency and impedance [1]. For this purpose the target structure is immobilized on the surface of a quartz biosensor. A profound knowledge of the molecular structure of the surface layers is required for its reliable application and the evaluation of the results. This has prompted us to develop a MALDI imaging technique for the two-dimensional mass spectrometric detection of the interaction partners. Two examples of specific interactions have been investigated: firstly concanavalin A, a lectin from jack bean (Convalaria ensiformis) binds α-D-mannopyranosidglycolipids; secondly biotin is inactivated by the hen’s egg protein, avidin. The quartz plate with the sample under investigation on its surface was washed with water to remove excess buffer salts and then placed in a recess of a modified MALDI target. Different matrices were tested for their applicability and placed on the surface using a thin layer preparation. A new airbrush method enabled a homogenous application of the matrix. MALDI mass spectra were recorded using a Voyager DEFig. 1: Scanning MALDI image of the letter “A”, PRO instrument (Applied Biosystems, prepared by pipetting a concanavalin A 2+ Weiterstadt, Germany). The biosensor ([M+2H] ~13 kTh) solution on a quartz sensor. surface was characterised by 50 x 50 points of a quadratic area resulting in a resolution of 1111 points per cm2. The 2500 mass spectra were analysed using a custom-developed software algorithm and visualised using mesh or contour plots (Fig. 1). The developed method is not limited to QCM crystals and can be adapted for the characterisation of other ionisable compounds on the surface of a range of materials. Literature [1] A. Hildebrand, A. Schaedlich, U. Rothe, R.H.H. Neubert, Journal of Colloid and Interface Science 249 (2002) 274.

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Poster P89 Rapid Drug Monitoring in Biological Matrices Using SPME // LC-MS/MS N. Scherzer1, P. Fuchs2, W. Miekisch2, J. Schubert2, H. Lord3, J. Pawliszyn3, B. Drewelow1, R.G. Mundkowski1* 1

Institute of Clinical Pharmacology, Centre in Pharmacology and Toxicology, University of Rostock, Schillingallee 70, D-18057 Rostock, 2Department of Anaesthesiology and Intensive Care, University of Rostock, 3Department of Chemistry, University of Waterloo *phone +49-381-494-5827, fax -5782, e-mail: [email protected]

Background: Pharmaceutical analyses need to meet minimum requirements in terms of reliability and safety. However, therapeutic monitoring of drugs is requested to deliver results rapidly as well, in order to allow for an instant adaptation of dosaging. Such “bedside monitoring” is particularly challenging for complex assays by LC-MS. Interfacing with LC requires suitable sample composition and should provide implementation into automated processing. Solid phase micro extraction (SPME) introduced new quality and speed to GC analysis. Recently, a new generation of fibre coatings has been developed to assess analytes in aqueous samples. This study was designed to compare LC-MS/MS assays for drug monitoring in blood based on SPME with polypyrrol fibres vs. conventional sample preparation. Two antibiotics were chosen as analytes, erythromycin, an established macrolide, and linezolide, a member of the new class of oxazolidinones. Blood samples were spiked in vitro or obtained from pig experiments. Methods: Performance of the fibres were first optimized in vitro using buffer and whole blood as adsorption media, various desorption solvents and at varied incbation times. For ex vivo experiments pigs were administered Erythromycin and Linezolid by intravenous infusion and heparinized blood samples were taken in a time range up to 120 minutes. Sample preparation was performed by SPME and conventional validated methods. Quantification of the analytes and the corresponding internal standards was carried out by LC-MS/MS. Results and Conclusions: The concentration vs. time curves determined by either SPME//LC-MS/MS or conventional analysis showed similar profiles for both erythromycin and linezolid, except the absolute concentration values which are generally higher for samples of SPME. Total analysis time is drastically reduced by SPME. As with GC, SPME appears to be a promising method for LC too. However, further work needs to be done on reproducibility, particularly with respect to unknown interferences caused by add-ons used for conventional sample preparations such as heparin or EDTA.

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Poster P90 Large-scale quantitative proteome analysis of apoptotic human cells using ICPL (Isotope-Coded Protein Label) and µLC-MALDI Alexander Schmidt, Cornelia Ciosto, Josef Kellermann, Friedrich Lottspeich Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Quantitative proteome analyses usually are accomplished by 2-dimensional electrophoresis (2DE) followed by mass spectrometric protein identification. Although this method is well established, accurate quantitative determination and high reproducibility of 2DE needs good expertise and experience. Here we describe a new method termed Isotope Coded Protein Label (ICPL) which overcomes many limitation of 2DE. The ICPL approach is based on isotopic labelling of all free amino groups in proteins. After multiplexing, any protein separation method may be adopted for reduction of complexity on the protein level (e.g. 1D, 2DE, free flow electrophoresis, liquid chromatography (LC), etc.) followed by cleavage, high throughput multidimensional LC and tandem mass spectrometry (MADLI-TOF/TOF). To demonstrate the effectiveness of this approach, a comparative proteome analysis of healthy and apoptotic human cells was performed. More than 400 proteins could be identified and quantified, resulting in the determination of more than 60 apoptotic regulated proteins. Furthermore, the method allowed the detection and quantification of over 25 posttranslationally modified protein species, including acetlyation, methylation and phosphorylation sites.

References: [1] Schmidt, A. et al; Proteomics, 2004, Dec 15, [ahead of print]

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Poster P91 Studying Calmodulin-Adenylyl Cyclase VIII Interaction with Chemical Cross-Linking and FTICR Mass Spectrometry Andreas Schmidt1, Daniela M. Schulz1, Stefan Kalkhof1, Christian Ihling1, Annette G. Beck-Sickinger3, Dermot M.F. Cooper2, and Andrea Sinz1 1

Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Linnéstraße 3, 04103 Leipzig 2 Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, United Kingdom 3 Faculty of Biochemistry and Pharmacology, University of Leipzig, Brüderstraße 34, 04103 Leipzig For structural studies of proteins and their complexes, a number of techniques, e.g. nuclear magnetic resonance spectroscopy (NMR), x-ray crystallography, and electron microscopy are employed. Just recently, chemical cross-linking combined with mass spectrometry has emerged as an alternative strategy to obtain low-resolution structural data of protein interfaces from low quantities of proteins within a relatively short time [1, 2]. In our present work, we study the interaction between the C-terminal region of adenylyl cyclase VIII and its binding partner calmodulin. Adenylyl cyclases are a critically important family of multiply regulated signaling molecules. Adenylyl cyclase VIII is regulated by Ca2+/calmodulin and possesses two possible calmodulin binding sites [3]. The N-terminal region exhibits a calcium-dependent calmodulin binding motif (amino acids 3554), whereas a calcium-independent IQ-motif [4] is located at the C-terminus (amino acids 1197-1210). A 25-amino acid peptide with amidated C-terminus comprising the IQ-motif was synthesized. The cross-linking reaction was performed by using different amine-reactive, isotopically labeled (d0 and d4) cross-linkers (BS3, bis[sulfosuccinimidyl]suberate and DSG, disulfosuccinimidyl glutarate) with varying spacer length and a zero-length cross-linker (EDC, ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride). The cross-linked 1:1 complexes were investigated by MALDI-TOF mass spectrometry to check the extent of chemical cross-linking. The separation of intramolecular and intermolecular cross-linking products was carried out by one-dimensional gel electrophoresis (SDS-PAGE). After excision of the bands of the cross-linked complexes, in-gel digestion was performed with trypsin resulting in complicated mixtures containing peptides modified by the cross-linker as well as intramolecular and intermolecular cross-linking products. These mixtures were analyzed by nano-RP-HPLC/ nano-ESI-FTICR mass spectrometry. We expect to gain further insight into the calmodulin-adenylyl cyclase VIII interaction based on the identified intermolecular cross-linking products. [1] Kalkhof S., Ihling C., Mechtler K., Sinz A., Anal. Chem., published online December 7, 2004 [2] Sinz A., J. Mass Spectrom., 2003, 38, 1225-1237 [3] Gu C., Cooper D.M.F., J. Biol. Chem., 1998, 274, 8012-8021 [4] Bähler M., Rhoads A., FEBS Letters, 2001, 513, 107-113

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Poster P92 Multidimensional HPLC In Proteomics Oliver Schmidt, Thomas Schulenborg, Christiane Lohaus, Helmut E. Meyer und Katrin Marcus Medical Proteome-Center, Ruhr-University Bochum

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is used as a common standard approach in proteomics. Nevertheless, its drawbacks (e.g. gel-to-gel variations, difficult handling and insufficient resolution of membrane-associated proteins) have in part limited its widespread use and application. Recently, various liquid-phase based separation techniques have been explored. The ability to characterise a complex protein sample (e.g. the brain proteome) by mass spectrometry (MS) depends on the power and sensitivity of the separation technique employed prior to the MS analysis. HPLC techniques are usually characterised by a high reproducibility paired with easy handling and a high grade of automation. Good results could be obtained by separating peptide mixtures using HPLC coupled with nano-LC-ESI-MS/MS as shown in several publications e.g.[1,2,3]. Unfortunately, tryptic digests of samples like brain tissue are too complex for an efficient differential proteome study using HPLC. One approach is the separation of the proteins prior any digestion, but the performance of HPLC techniques is insufficient when using brain tissue. The high complexity of such samples and the large dynamic range needed when analysing complex protein mixtures demands a multi-dimensional separation technique. In our lab we separated and analysed the Serva Proteome Standard (a mixture of 8 proteins) for optimisation of the system as well as mouse brain lysate with both approaches 2DPAGE and 2D-HPLC, respectively. The proteins were identified by nano-LC-ESI-MS/MS after tryptic in-gel or in-solution digestion. In practice, HPLC based separation of these samples as well as 2D-PAGE approaches have some challenges: 1. handling of hundreds of fractions (off-line setups) 2. very time consuming 3. insufficient quantification. Furthermore, convenient software to visualise and interpret HPLC data for a differential proteome study is still missing. In future some improvements on the HPLC techniques are needed, to enhance their performance and to allow quantification to make differential proteome studies possible.

[1] Wang et al., Journal of Chromatograpy B, 787 (2003) 11-18 [2] Smith et al., Journal of Proteome Research, 3 (2004) 68-75 [3] Le Bihan et al., J. Am Soc Mass Sectrom 14 (2003) 719-727

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Poster P93 Massenspektrometrische Untersuchungen zur Bildung von Polymeren in atmosphärischen sekundären organischen Aerosolen Mathias Schott, Jörg Warnke, Thorsten Hoffmann Institut für Anorganische und Analytische Chemie der Johannes Gutenberg-Universität Mainz, Duesbergweg 10-14, 55128 Mainz Atmosphärische Aerosole wirken direkt und indirekt auf die Strahlungsbilanz und damit auf das Klima der Erde, da sie z.B. das Sonnenlicht streuen und als Wolkenkondensationskeime dienen können. Ebenso können Aerosole die Gesundheit des Menschen beeinflussen. Die feine Fraktion (Durchmesser um 100 nm) der atmosphärischen Aerosole besteht hauptsächlich aus sekundären Aerosolen. Sekundäres Aerosol entsteht, wenn leicht flüchtige Vorläufersubstanzen in der Atmosphäre durch chemische Reaktionen (z. B. Oxidation) in schwer flüchtige Produkte umgewandelt werden. Diese Produkte können durch Kondensation auf vorhandene Partikel oder sogar durch Bildung neuer Partikel zur Bildung sekundärer Aerosolpartikel führen. Sekundäre organische Aerosole (SOA) entstehen aus leicht flüchtigen organischen Vorläufersubstanzen biogenen oder anthropogenen Ursprungs. Die Zusammensetzung und Herkunft des organischen Anteils atmosphärischer Aerosolpartikel ist jedoch trotz einiger Anstrengungen nur zum Teil geklärt. Für den größten Teil des SOA werden Monoterpene, ungesättigte C10 Kohlenwasserstoffe, verantwortlich gemacht. Bisher standen dabei die Produkte der Ozonolyse oder der Reaktion mit OH-Radikalen im Vordergrund des Interesses, die vor allem durch Carbonsäurefunktionen charakterisiert waren. Neuere Untersuchungen deuten jedoch auch auf Polymerisationsreaktionen in der Partikelphase hin. Dabei könnten mittelflüchtige Carbonylfunktionen tragende Oxidationsprodukte in schwerflüchtige Substanzen mit hohem Molekulargewicht umgewandelt werden [1,2,3]. Dies würde die Bilanz der Bildung schwer flüchtiger Substanzen aus den leicht flüchtigen Kohlenwasserstoffen wesentlich beeinflussen, sowohl in Bezug auf biogene als auch auf anthropogene Kohlenwasserstoffe. Obwohl die bisherigen Untersuchungen gute Anstöße für eine weitere Forschung liefern, sind die durchgeführten massenspektrometrischen Charakterisierungen der Reaktionsprodukte oft unzureichend für eine eindeutige Aussage. Daher soll durch eine systematische Erforschung von möglichen Polymerisationsreaktionen in der Partikelphase durch massenspektrometrische Methoden wie LC-ESI-MS/MS oder online-APCI-MS/MS die Bildung schwerflüchtiger Produkte aus mittelflüchtigen Oxidationsprodukten bzw. direkt aus der Ozonolyse oder Photooxidation von Kohlenwasserstoffen untersucht und bewertet werden. Für die Simulation der Bildung von SOA im Labor wird eine Glaskammer benutzt, in der leichtflüchtige Substanzen mit Ozon unter Lichteinstrahlung zur Reaktion gebracht werden können. In ersten Schritten können jedoch die Polymerisationsreaktionen mit einfachen Monomeren durchgeführt werden, damit Informationen zu den Reaktionsmechanismen und den Reaktionsbedingungen in überschaubaren Modellsystemen erhalten werden können. Literatur [1] [2] [3]

M. Kalberer, SCIENCE, Vol 303 (2004), 1659-1662 M. P. Tolocka, Environmental Science & Technology, Vol 38 (2004), No. 5, 1428-1434 Y. Iinuma, Atmospheric Environment, 38 (2004), 761-773

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Poster P94 Protein and Phosphoprotein Analysis Using a 4000 Q Trap mass spectrometer Thomas Schulenborg1, David Fischer2, Helmut E. Meyer1, Fred van Leeuwen2, Katrin Marcus1 1 2

Medical Proteom-Center, Ruhr-University Bochum Netherlands Institute for Brain Research, Amsterdam

Phosphorylation is a very prominent post-translational modification (PTM) of proteins and plays an important role in regulation of many cellular processes, folding, localisation and the interaction of proteins [1]. In most instances phosphorylations emerge as transient modifications within signalling cascades and are accompanied by dephosphorylation. Hence, the absolute quantity of phosphorylated protein species is less than 1% of the whole protein amount. Assuming that one 2D-gel spot contains about 1-10 pmol protein, just 10100 fmol phosphorylated species is available for the proteomic analysis. In consequence, the identification and localisation of phosphorylation sites is quite difficult, thus often an enrichment of the phosphoproteins/-peptides prior to the analysis is required. Indeed, every additional step in sample preparation holds the risk of incalculable protein loss. In the present work we will demonstrate the necessity of extensive optimisation work in phosphoproteomics and related to this the power of quadrupole/linear ion trap mass spectrometers in the identification and localization of phosphorylation sites without prior enrichment of the phosphorylated proteins. In a 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) based on differential phosphoproteome study of a Alzheimer’s mouse model (ubiquitin+1, UBB+1 transgenic mice) [2, 3] a hybrid triple quadrupole/linear ion trap mass spectrometer – the 4000 QTrap® (Applied Biosystems) will be used for the nanoLC-MS/MS analysis of phosphorylated proteins. For evaluation of the instrument different standard proteins and phosphoproteins were analysed, such as alphaA-Crystallin [4, 5, 6], protein kinase A and LASP. The proteins were either tryptically digested in solution or protein separation was done using 2D-PAGE or 1D-PAGE followed by in-gel digestion. After extensive optimisation, including solvent composition, gradient time, and scan types etc., the sequence coverage of the individual protein and at the same time the number of detected and localised phosphorylation sites could be tremendously increased. .

Literatur [1] Kurosawa, M, J Pharmacol Toxicol Methods, 1994, 31, 135-9 [2] Fischer et al., FASEB J, 2003, 17, 2014-4 [3] van Leeuwen et al., Science, 1998, 279, Issue 5348, 242-7 [4] MacCoss, MJ et al., Proc Natl Acad Sci U S A. 2002, 99, 12, 7900-5 [5] Schaefer, H et al., Anal Bioanal Che, 2003, 376, 7, 966-72 [6] Hogan JM et al., Anal Chem. 2003, 75, 23:6509-16

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Poster P95 Purification and Characterization of the Tetrameric Complex Between Annexin II and p11 from Porcine Small Intestine Daniela M. Schulz 1, Andreas Schmidt 1, Olaf Zschörnig 2, Stefan Kalkhof 1, Christian Ihling 1, and Andrea Sinz 1 1

Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Linnéstr.3, 04103 Leipzig, Germany 2 Institute for Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, 04107 Leipzig, Germany

The annexin II / p11 heterotetramer (A2t) is a multifunctional protein composed of two annexin II and two p11 (S100A10) subunits. Ca2+-binding mediates its interaction with phospholipids in membranes. A2t plays an important role in membrane interactions as well as membrane trafficking and signaling [1].The purified tetrameric complex is intended to be used for structural studies of the annexin II / p11 interface. Annexin II / p11 tetramer (A2t) was purified from pig (Sus scrofa domestica) small intestine and the constituent components were characterized by nano-HPLC/nano-ESI-FTICR mass spectrometry as well as by MALDI-TOF mass spectrometry. The purification protocol was adapted from Gerke and Weber [2] and slightly modified. Briefly, mechanically disrupted mucosal scrapings from porcine small intestine were washed repeatedly with buffer and centrifuged, retaining the pellet with A2t bound to the membrane in the presence of calcium. Addition of EGTA and subsequent ultracentrifugation released the tetrameric complex from the membrane into the supernatant. Further purification was achieved by ion exchange chromatography. A2t was found to be about 90% pure (based on SDS-PAGE) after the first chromatography step. Both the monomers and the impurities were in-gel digested by trypsin, AspN, and LysC and analyzed by ESI-FTICRMS and MALDI-TOFMS. Sequence coverages from peptide mass fingerprinting of annexin II and p11 were 100% and 72%, respectively. Several other annexins were identified from samples collected during the purification process. Our data on the sequence of porcine annexin II confirm the recently published amino acid sequence [3] in the NCBI database. The purified A2t complex will be characterized with respect to its three-dimensional structure employing chemical cross-linking and FTICRMS as described previously [4]. References [1] Gerke V. and Moss S. E. (2002), Physiol. Rev. 82: 331-371 [2] Gerke V. and Weber K. (1984), EMBO J. 3: 227-233 [3] Danielsen E. M., van Deurs B., and Hansen G. H. (2003), Biochemistry 42: 14670-14676 [4] Schulz D. M., Ihling C., Clore G. M., and Sinz A. (2004), Biochemistry 43: 4703-4715

202

Poster P96 Enrichment of glycoproteins and glycopeptides supported by magnetic particles and detected by MALDI-TOF-MS Katrin Sparbier, Sonja Koch, Irina Keßler, Thomas Wenzel, Markus Kostrzewa Bruker Daltonik GmbH, Permoserstr. 15, 04318 Leipzgi, [email protected]

Glycosylation is with at least 50 to 80 percent the most common form of post-translational modification of proteins. Since it turns out that the degree and the type of glycosylation depends on the status quo of cells and is mostly linked to certain diseases the discovery and identification of those modified peptides and proteins gain more and more importance in diagnosis. In particular aberrant or missing glycosylation can be utilized as profiling parameter. MALDI-TOF mass spectrometry is an ideal technique for identifying a large number of peptides and proteins and their corresponding modifications. Enrichment, isolation and purification of glycosylated peptides and proteins from different sources - a prerequisite for MALDI-TOF mass spectrometry - can be attained by affinity chromatography supported by magnetic particles. Concanavalin A (ConA) and di-boronic acid functionalized magnetic particles were developed to enrich glycosylated peptides and proteins. ConA specifically binds mannosyl and glucosyl residues of polysaccharides and glycoproteins [1] containing unmodified hydroxyl residues at positions C3, C4 and C6 [2] and can be applied as a general tool for capturing of N-glycosylated peptides and proteins. In contrast, boronic acid forms a covalent bond with 1,2-cis-diol group containing molecules [3] like mannose, galactose or glucose. Thereby it additionally facilitates the enrichment of the more heterogeneous Olinked oligosaccharides but not the capture of N-linked oligosaccharides of the complex type. The functionalized beads were employed to establish and optimize protocols for the binding and detection of glycosylated peptides and proteins with respect to an automated workflow and the subsequent detection and identification by MALDI-TOF mass spectrometry. For several model proteins the capture could be demonstrated by SDS-PAGE and MALDI-TOF mass spectrometry. According to the type of glycosylation, high mannose, hybrid or complex type, the different proteins were enriched by ConA or boronic acid functionalized beads, respectively. RNase B could be isolated from spiked human serum samples by ConA beads according to the well-known N-linked high mannose oligosaccharide structure. References [1] Goldstein IJ, Hollerman CE, Smith EE. Protein-Carbohydrate interaction. II. Inhibition studies on the interaction of Concanavalin A with polysaccharides. Biochemistry. 1965 May;41:876-83. [2] Yahara I, Edelman GM. Restriction of the mobility of lymphocyte immunoglobulin receptors by Concanavalin A. Proc Natl Acad Sci U S A. 1972 Mar;69(3):608-12. [3] Rawn JD, Lienhard GE. The binding of boronic acids to chymotrypsin. Biochemistry. 1974 Jul 16;13(15):3124-30.

203

Poster P97 Ethylenediamminedichloroplatinum and Cisplatin: A Study by Electrospray Ionization - High Resolution Mass Spectrometry A. Springer1 and M. Linscheid1 1

Humboldt Universität zu Berlin, Department of Chemistry, Analytical & Environmental Chemistry, Brook-Taylor-Str.2, 12489 Berlin.

More than 40 years after the discovery of the antitumor activity of Cisplatin (cisPt(NH3)2Cl2, cDDP) by Rosenberg it is still one of the most important substances in cancer treatment, and it is likely the best known one. Strong side effects and a rapid development of resistance against its mode of action are the greatest troubles which have been tried to overcome with modifications of the precursor agent. But due to its ability as a broadband antitumor drug cisplatin is still in use, even with more than 1000 analogues synthesized [1]. Several years and some hundred publications later, its mode of action still is not fully understood. Maybe more information about the drug and its analogues can be of some help to circumvent the time and resource wasting trial-and-error principle used until now. With the life span of humans increasing steadily the interest in cancer treatment will even be one of the biggest future challenges. A systematic study by ESI-LIT/FTICR MS of ethylendiamminedichloroplatinum is presented. High resolution data are represented to prove the elemental compositions of the major ions. Cisplatin and its Electrospray-generated ions have been published lately [2], [3], but some new insights are presented on this poster. We were able to confirm the structures of ions described previously measured with low-resolution mass spectrometers with high resolution experiments. Structures and elemental compositions of ions of formerly unknown composition have been elucidated. Some ions not described before and their fragmentation pathways in linear ion traps with FTICR detection are presented. Literatur [1] B. Lippert (Ed.) Cisplatin: chemistry and biochemistry of a leading anticancer drug, Wiley-VCH (Weinheim), 1999 [2] M. Cui and Z. Mester Rapid Commun. Mass Spectrom. 2003, 17, 1517-1527 [3] M. Cui, L. Ding and Z. Mester Anal. Chem. 2003, 75, 5847-5853

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Poster P98 Biogenic iodine compounds in environmental samples and their determination by GC-MS Nicola Springer 1, Thorsten Hoffmann 1 1

Institute of Inorganic and Analytical Chemistry, Gutenberg University Mainz, Duesbergweg 10-14, D-55128 Mainz

In literature the role of organic and inorganic iodine compounds in the marine boundary layer has been discussed for several years now. CH2I2 is said to be a key compound, it can be detected in seawater as well as in marine air. Macroalgae as well as Phytoplankton can synthesize this and other compounds from iodine precursors and release them to the atmosphere. Under UV-radiation Diiodomethane can break down to iodine radicals which can react with ozone to iodine oxides [1]. Those oxides are associated with new particle formation in the marine atmosphere [2]. The formation of aerosols is of high interest because the particles have a large influence on the climate. Depending on their composition they can have direct or indirect effect on the Earth´s radiative balance as well as on human health [3]. Therefore several different methods are used to analyse iodine species in different matrices (water, particles, air). There are several possibilities analysing elementary iodine. One is to use element-analytical methods such as ICP-MS, after denuder sampling and extraction. However, iodine species in the aqueous or particle phase (e.g. iodine, iodide and iodate) can also be derivatised to a volatile organic compound. Then quantification can be made by GC iontrap-MS. The analytical methodology presented here is based on the reaction with N,Ndimethylaniline to form 4-iodo-N,N-dimethylaniline. The reaction conditions can be chosen to derivatise only iodine in the presence of iodide and iodate. Iodide can be determined as a sum parameter with iodine. The iodate concentration can finally be calculated from the total iodine concentration [4]. 3 HSO3- + IO3-  I- + 3 HSO4I

OI

+

-

+

Na + 2 I + 2 H 

+

COO-

Na + I2 + H2O

COON

N

+ I2 

+ HI I

[1] L.J. Carpenter, Chemical Reviews, Vol 103, 4953-4962, 2003 [2] C. O`Dowd, Nature, Vol 417, 632-636, 2002 [3] C.E.Kolb, Nature, Vol 417, 597-598, 2002 [4] S. Mishra, The Analyst, Vol 125, 459-464, 2000

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Poster P99 A nano electrospray ionization - ion mobility spectrometer coupled to ion trap mass spectrometry for fast separation and structure elucidation of dendrimers and their by-products Ludwig Schwarz 1, and Michael Linscheid 1 1

Institut für Chemie, Humboldt-Universität zu Berlin

An in-house built nano electrospray ionization ion mobility spectrometer (IMS) was used to separate on a millisecond time scale a poly(propyleneimine) dendrimer and its synthesis byproducts. A coupled ion trap mass spectrometer allowed for sampling of ions from several IMS scans, mass spectrometric identification of separated species and fragmentation (MS^n) for further structure elucidation [1], [2], [3]. Drift times of singly and multiply protonated dendrimer molecules were determined from which collision cross sections were calculated. A strong increase in collision cross sections with increasing charge number shows that upon successive protonation the molecule unfolds from a compact conformation to more and more extended conformations, driven by Coulomb repulsion between charged sites. Literatur [1] S. McLuckey, K. Asano, T. Schaaff, and J. Stephenson Int. J. Mass Spectrom. 2000, 195/196, 419-437 [2] J. Weener, J. van Dongen, and E. Meijer J. Am. Chem. Soc. 1999, 121, 10346-10355 [3] J. Hummelen, J. van Dongen, and E. Meijer Chem. Eur. J. 1997, 3, 1489-1493

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Poster P100 Photoactivities of coated TiO2 in aqueous and water-oil system measured by HPLC-Ion trap mass spectrometry Guangzu Zhang , Michael Linscheid Department of Chemistry, Humboldt Universität zu Berlin, Brook-Taylor-Str. 2, D-12489

Skin cancer is one of the most common cancers in the world now a day. Sunscreens are widely used to protect our skin from irradiation in the sun light. TiO2 does not absorb radiation in the visible region of the spectrum. It reflects and scatters UVB and UVA in the sunlight. Thus TiO2 is a common ingredient of sunscreens and cosmetics. However, TiO2 is known to be photobiologically active when irradiated with UVA. When suspension of TiO2 is irradiated with UV light at wavelength shorter than 385nm, TiO2 can be excited and generate reactive oxygen species (1O2, O2-, .OOH). These reactive oxygen species can attack biomolecules including DNA [1,2] and cause damages. Since TiO2 shows certain photoacitivies, it may be a potential danger to use it without treatment in sunscreens. For this reason, more and more coated TiO2 materials are used in sunscreens instead of uncoated TiO2. 8-oxodG is generally considered to be an indicator of oxidative DNA damage. An analytical method was developed to quantify 8-oxodG in calf thymus DNA in our lab. Our results about the measurement of 8-oxodG generated in UVA irradiated DNA using TiO2 as photosensitizer by HPLC-Ion trap mass spectrometry was presented in last year’s Annual meeting DGMS. In this work, the photoactivities of two coated TiO2 materials were measured as 8-oxodG level in UVA-irradiated DNA against UVA doses. In the end the photoactivities of coated TiO2 materials were compared with those of uncoated TiO2 and without TiO2. Literatur 1. Ashikaga, T., et al., Effect of the photocatalytic activity of TiO(2) on plasmid DNA. Mutat Res, 2000. 466(1): p. 1-7. 2. Wamer, W.G., J.J. Yin, and R.R. Wei, Oxidative damage to nucleic acids photosensitized by titanium dioxide. Free Radic Biol Med, 1997. 23(6): p. 851-8.

207

Poster P101 Mass spectrometric studies of the maleonitrile tetrathia crown ethers and their complexes with different transition metal ions I. Starke, S. Fürstenberg, H. Müller, H.-J. Holdt, E. Kleinpeter Department of Chemistry, University of Potsdam, P.O. Box 60 15 53, D-14415 Potsdam, Germany Maleonitrile- dithiacrown ethers are well known as preorganized S2On (n=2-5) coronands which force B, AB and A class metal ions into mixed S/O coordination spheres. Moreover, they form chelate complexes with MX2 salts (M=Pd, Pt; X=Cl, Br).1 Along this study the complex formation behaviour of the maleonitrile tetrathia crown ethers mn12S4 and mn13S4 (depicted in Figure 1) towards different transition metal salts (MX2, M=Hg, Pd, Pt; X=Cl, J) was studied by Electrospray Ionization and MS/MS experiments. mn12S4 NC

NC

S

S

S

S

mn13S4 NC

S

S

NC

S

S

Figure 1

Energy-variable collisional activation is used to promote the fragmentation of the maleonitrile ligand / metal complexes which are of different complex stoichiometry. Both [2 : 1] and [1 : 1] complexes with silver(I) salts are observed in the mass spectra with different intensities. The fragmentational behaviour of these [2 : 1] and [1 : 1] complexes with AgY (Y=BF4, ClO4 and PF6) were studied. In case of the silver complexes, increasing collisional energy lowers the relative abundance of the complex ion peak [2 : 1] +, but raiser the abundance of the complex ion peak of [1 : 1]+. CID measurements were carried out to compare the stability of the [2 : 1] and [1 : 1] complexes. The activation voltages for the dissociation of the complexes with silver (I) were generelly greater in the [1 : 1] complexes than in the corresponding [2 : 1] complexes. In the CID experiments of the free maleonitrile tetrathia crown ethers the fragmentation occurred similarly in both ligands, mainly involving cleavage of the bonds between the sulfur atoms and the carbons attached to the ethylene unit. Common fragment ions, which correspond to the losses of CH2-CH2, CH2CH2-S, CH2CH2-S-CH2CH2 moieties, are observed. Differences in the fragmentational behaviour of the free crowns and of their different [metal : ligand] complexes will be discussed in detail. The ESI spectra were recorded using a Micromass Q-TOFmicro mass spectrometer in positive electrospray mode. For MS/MS, after selection of the appropriate precursor ion, argon was used as the collision gas (~ 5 x 10-5 mbar) and the gas cell was maintained between 2 - 40 eV. Elemental compositions for the fragments were determined by accurate mass measurement with standard deviation < 10ppm. Literatur [1] H.-J. Drexler, I. Starke, M. Grotjahn, E. Kleinpeter, H.-J. Holdt, Inorg. Chim. Acta 317, (2001) 133-142

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Poster P102 LC-MS/MS in der Rückstandskontrolle – Kokzidiostatika in Eiern Michael Stephan1, N. Palinsky2, M. Ludewig2 1

Landesamt für Verbraucherschutz Sachsen Anhalt, FB Lebensmittelsicherheit, Freiimfelder Straße 66-68, 06112 Halle (Saale) 2 Universität Leipzig, Institut für Lebensmittelhygiene, An den Tierkliniken 1, 04103 Leipzig Ökologisch erzeugte Lebensmittel nehmen gegenwärtig an Bedeutung zu. Ihr Marktanteil ist in den letzten Jahren kontinuierlich gestiegen; gegenüber dem Jahr 2001 hat die ökologisch bewirtschaftete Fläche in Deutschland nach Angaben des Bundesministeriums für Verbraucherschutz, Ernährung und Landwirtschaft (BMVEL) im Jahr 2002 um 9,8% zugenommen und damit einen Anteil von 4,1% der gesamten landwirtschaftlichen Nutzfläche. Da der Aufschwung der Ökoerzeugung erst von relativ kurzer Zeitdauer ist, liegen bislang nur wenige wissenschaftliche Ergebnisse über die Qualität tierischer Erzeugnisse aus ökologischem Landbau vor. In einem Forschungsprojekt des Instituts für Lebensmittelhygiene der Universität Leipzig wurden im Rahmen des Bundesprogramms „Ökologischer Landbau“ Analysen zur Qualität von Eiern durch-geführt. Gesundheitliche Unbedenklichkeit von Lebensmitteln ist nicht nur gekennzeichnet durch das Freisein von pathogenen Erregern, sondern auch durch die Unterschreitung von Rückstandshöchstmengen eingesetzter Arzneimittel. In dem hier dargestellten Teilprojekt wurden jeweils 100 Proben ökologisch und konventionell produzierter Hühnereier auf die Vorkommen der Polyether-Antibiotika Maduramycin, Narasin, Monensin, Lasalocid und Salinomycin sowie des Wirkstoffs Nicarbazin untersucht. Die als Kokzidiostatika bezeichneten Polyether und auch Nicarbazin sind gegen einzellige Darmparasiten (Kokzidien) bei Geflügel gerichtete Wirkstoffe. Der Einsatz dieser Stoffe als Arzneimittel ist nach EU-Verordnung (EWG) Nr. 2377/90 des Rates in der Legehennenhaltung nicht zugelassen. Nicarbacin, Maduramycin und Narasin sind auch nach der FuttermittelVerordnung (2000) als Futtermittelzusatzstoffe in der Hennenhaltung nicht erlaubt. Dagegen dürfen nach EU-Richtlinie 70/524/EWG (2000) Salinomycin bis zur 12. Lebenswoche, Monensin und Lasalocid bis zur 16. Lebenswoche als Zusatzstoffe in Futtermitteln zum Einsatz bei Mastgeflügel, Junghennen und Truthühnern verabreicht werden. Zur quantitativen Bestimmung der genannten Arzneimittel in Ei wurde die Probenaufbereitung modifiziert und eine LC-ESI-MS/MS-Methode etabliert und validiert, die eine selektivere und um mindestens Faktor 10 sensitivere Alternative zur bisher gebräuchlichen HPLC-Methode mit Nachsäulenderivatisierung und Detektion mittels UV/VIS bzw. FD darstellt. Die Wiederfindungen der hier vorgestellten Methode betragen für die analysierten Stoffe zwischen 21% und 54%. Es wurden Reproduzierbarkeiten zwischen 11% und 28% gefunden. Für die nach der Entscheidung der Kommission 2002/657/EG betreffend die Durchführung von Analysemethoden und die Auswertung von Ergebnissen erforderlichen Bestimmung der Leistungsgrenzen der Methode wurden für die Entscheidungsgrenze CC (Grenzwert, bei und über dem mit einer Fehlerwahrscheinlichkeit von =1% bestimmt werden kann, dass eine Probe positiv ist) und das Nachweisvermögen CC (Kleinster Gehalt eines Stoffes, der mit einer Fehlerwahrscheinlichkeit von =5% in einer Probe nachgewiesen und/oder quantifiziert werden kann) Werte von CC=0.35 µg/kg und CC=0.55 µg/kg für die Polyether bzw. CC=0.03 µg/kg und CC=0.04 µg/kg für Nicarbazin ermittelt. 209

Poster P103 SARAMIS A NEW WAY OF DATA MANAGEMENT OF MALDI-TOF MS FOR MICROORGANISM-IDENTIFICATION M. Erhard1, W. Kallow1 and S. Sauermann1 1

AnagnosTec, Gesellschaft für Analytische Biochemie und Diagnostik mbH

Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Mass Spectrometry (MALDITOF MS) for classification and identification of microorganisms became more and more popular during the last years. Our company has developed a new software in order to handle the enormous volumes of data and to make the analysis of the results fast and easy: SARAMIS (Spectral Archiving And Microbial Identification System) MALDI-TOF MS data are reduced to easy-to handle mass lists and imported to the database. The Comparison of MS-data followed by a patented procedure that generates theoretical spectra (superspectra) enables the identification of various classes of microorganisms. Having recorded specific MS-signals of family, genus, species and strain, these signals are then ranked according to their specificity. High quality superspectra can be used for automatic identification of family, genus, species or even strain level. In case of unknown organisms the superspectra allow grouping and dereplication of samples. As in sequence and RNA analyses dendrograms are also calculated, here basing on the specific characteristics of MALDI-TOF MS data. This is done twofold: First method is based on the acquired absolute mass matches and second taking in account the percentage identity of spectra. By using a special export routine and file format all MALDI-TOF MS-data can be imported into SARAMIS, regardless to the manufacturer of the MALDI-TOF hardware.

210

Poster P104 Gas-phase chemistry of 1,2,3,4-tetrahydrobenzopyran-2,3-dicarboxylic acid derivatives upon electron ionization by means of metastable mass spectrometry R. Szmigielski, W. Danikiewicz, , K. Dolatowska, K. Wojciechowski Institute of Organic Chemistry, Polish Academy of Sciences, ul. Kasprzaka 44/52, 01-224, Warszawa, Poland, e-mail: [email protected] Although metastable mass spectrometry with electron ionization source has been known for years, this technique up to now constitutes a good tool for study of the gas-phase chemistry of various organic compounds. It provides with information not only concerning the fragmentation pattern of compounds of interest but also gives insight into the mechanisms of observed unimolecular reactions of ions. Moreover, in many cases it enables to assign the structure of ions in the gas phase that is challenge of its own especially for chemists linking in their work skills in mass spectrometry and organic chemistry [1]. In our poster we wish to show the basic fragmentation patterns for cis-9,9a-dihydro3aH-4-oxa-2-azacyclopenta[b]naphthalene-1,3-dione derivatives (1) 3 O which are formed in solution by the reaction of highly reactive, nonR X isolable methylenequinone-type intermediates with N-substituted 2 N R phenylmaleimides. It was found that upon electron ionization the 1 fragmentation starts at the succinimide ring and its pattern depends R H O on the substituents at the carbon atom position 5-8. It is interesting to 1 X=O note that peaks which can be assigned to the product of the retro 2 X = N-CH3 Diels-Alder opening (RDA) of the nitrogen atom containing tetrahydrobenzopyran ring were also observed. However, in comparison to our provious results [2], obtained for the structural analogue (2), the RDA process for compounds under study (1) results in the retention of a charge at phenylmaleimide ring. Literature [1] H. Budzikiewicz H, R. D. Grigsby "Half protons or doubly charged protons? The history of metastable ions", J.Amer. Soc. Mass Spectrom., 15 (9): 1261-1265 (2004). [2] W. Danikiewicz, K. Wojciechowski, “Fragmentation of 1-methyl-1,2,3,4-tetrahydroquinoline-2,3dicarboxylic acid derivatives upon electron ionization”, Pol. J. Chem. 72: 554-563 (1998).

211

Poster P105 Identification of toxicologically relevant proteins from rat liver Kareen Tenz1,2, Stephanie Lamer1, Axel Oberemm2, Michael Schümann1, Eberhard Krause1 1

Institute of Molecular Pharmacology, D-13125 Berlin, Germany; Federal Institute of Risk Assessment, D-14195 Berlin, Germany

2

Proteomic approaches are widely explored in short term toxicological tests and for gaining insight into toxic and carcinogenic effects of chemicals. The analysis of global protein expression in rat liver in the time course of chemical exposure by the use of 2-DE based proteomics allows the detection of treatment- and process-related changes and provides insight into the mechanisms behind the toxic response. Gel-separated proteins were identified using peptide mass fingerprinting (PMF) and tandem mass spectrometry. Available protein information, i.e. protein identity, molecular weight, isoelectrical point and EC-number were combined into an HTML-document; identified proteins were grouped and plotted on a representative gel image. Protein identifications were performed via PMF and tandem MS using MALDI-TOF-MS (Voyager-DE STR, Applied Biosystems) and a nano-LC-ESI-quadrupole-TOF-MS (Q-TOF Ultima, Waters), respectively. Database search was performed using the Mascot search engine. The identified proteins could associated to different protein classes, for instance transcription factors, heat shock proteins, metabolic enzymes and detoxification enzymes. An important enzyme of the latter class is Glutathione S-transferase P, which is already described as a biomarker for cancerogenesis. The data shows that the expression of this enzyme correlates with the time course of exposure. In summary, the results show that the approach is suitable for the identification of many toxicologically relevant proteins and the detection of sub-chronic, low-dose effects of chemicals and xenobiotics. The PMF method allows a higher throughput of samples. The nano-LC tandem MS analysis is more time consuming, however, it shows a higher sensitivity and leads to more reliable protein ID’s. By combination of both methods we had identified more than 600 Proteins up to now. The recently established MALDI-TOF/TOF will combine the confidence of MS/MS results with the high efficiency of the MALDI technology.

212

Poster P106 Pyridinium based ionic liquid matrices for the analysis of peptides by MALDI-ToF mass spectrometry Masoud Zabet-Moghaddam, Elmar Heinzle & Andreas Tholey Technische Biochemie, Universität des Saarlandes, 66123 Saarbrücken Since its introduction in the late 1980s, MALDI mass spectrometry became an indispensable tool for the analysis and identification of peptides and proteins. In the so called peptide mass fingerprint analysis proteins are hydrolyzed at defined cleavage sites using specific proteases. The resulting peptide spectra (fingerprints) are compared with in-silico-digests (database search) and highest matches can lead to the identification of the protein. A number of methods have been described to increase the quality of the peptide spectra in terms of sensitivity, reduction of unwanted adducts [1] as well as of the homogeneity of the sample preparations. In recent years, ionic liquid matrices (ILM) composed of equimolar mixtures of classically used crystalline MALDI matrices (e.g. DHB, CCA, SA) with organic bases [2,3] have been described to increase sample homogeneity, thus allowing a facilitated qualitative and quantitative analysis by MALDI MS [3-5]. In the present work the performance of mixtures of the matrix α-cyano-4-hydroxy-cinnamic acid (CCA) with pyridine (Py) for peptide (mass fingerprint) analysis was tested. Using mixtures of 5 standard peptides, we investigated the influence of different molar ratios between matrix and pyridine on signal intensities and signal-to-noise ratios. A molar ratio of 1.6:1 (CCA:Py) turned out to be optimal. No additional adducts of the analytes with pyridine were observed, peak shapes, mass accuracy and resolution of the signals were unaffected. The optimized CCA-Py-ILM was used for the analysis of tryptic protein digests (in solution- as well in gel-digests) of model proteins. The signal to noise ratios of the majority of the tryptic peptides was increased in case of the pyridinium bases ILM compared with pure CCA. In some cases weak peptide signals (in CCA) could be amplified in the ILM thus leading to a higher sequence coverage and thus to a facilitated identification of the proteins by database search. References [1] Smirnov, I.P, Zhu, X., Taylor, T., Huang, Y., Ross, P., Papayanopoulos, I.A., Martin, S.A. & Pappin, D.J. (2004). Anal. Chem., 76, 2958-2965. [2] Armstrong, D.W., Zhang, L.K., He, L. & Gross, M.L. (2001). Anal. Chem., 73, 3679-3686. [3] Zabet-Moghaddam, Heinzle, E. & Tholey, A. (2004). Rapid Commun. Mass Spectrom., 18, 141-148. [4] Bungert, D., Bastian, S., Heckmann-Pohl, D.M., Giffhorn, F., Heinzle, E. & Tholey, A. (2004). Biotechnol. Lett., 26, 1025-1030. [5] Li, Y.L. & Gross, M.L. (2004). J. Am. Soc. Mass. Spectrom., 15, 1833-1837.

213

Poster P107 Entwicklung eines quasi-kontinuierlichen Messverfahrens zur Überwachung von Vergasungsanlagen mit dem Ziel der Teerminderung Schulz-Tönnies, Karen 1, Neubauer, York 1, Behrendt, Frank 1 1

TU Berlin, Institut für Energietechnik, FG Energieverfahrenstechnik und Umwandlungstechniken regenerativer Energien.

Bei der Holzvergasung zur Erzeugung von H2-/CO-haltigem Brenngas (Nutzgas) entstehen je nach Betriebsbedingungen und Anlagen teerartige Kohlenwasserstoffe in verschiedenen Mengen und Zusammensetzungen. Werden diese Teere nicht vollständig entfernt, kommt es zu starken Verschmutzungen nachgeschalteter Anlagenteile. Ein kontinuierlicher Anlagenbetrieb wird dadurch stark eingeschränkt. Die Verbesserung der Gasqualität sowie die analytische Erfassung der Teerkomponenten sind Gegenstand der derzeitigen Forschung auf dem Gebiet der Vergasung. Bisher existieren weder eine einheitliche Definition der Teere noch gibt es standardisierte Messverfahren zur Bestimmung des Teergehaltes. Einige Forschungsgruppen bedienen sich eigener Methoden, meist mit aufwändigen und zeitintensiven nasschemischen Aufbereitungsschritten. Die Untersuchungen des Einflusses einzelner Betriebsparameter auf die Teerbildung ist mit den bekannten Methoden sehr aufwändig, da jeder Messpunkt mehrere Stunden gehalten werden muss, die Optimierung des Vergasungsprozesses wird somit langwierig und kostenintensiv. Ein aktuelles, durch die Fachagentur Nachwachsende Rohstoffe gefördertes Projekt beinhaltet den Aufbau einer Wirbelschichtvergasungsanlage zur Lieferung des Brenngases, den Aufbau der katalytischen Versuchsapparatur zur kostengünstigen Entfernung von Teer und anderen Partikeln aus vergaster Biomasse sowie ein neu zu entwickeltes Lasermassenspektrometer zur Analyse der Teerverbindungen. In diesem Beitrag werden die Entwicklung eines quasi-online- Messverfahrens für den Gehalt an polyzyklischen aromatischen Kohlenwasserstoffen im Gas aus der Biomassevergasungsanlage sowie dazu notwendige Umbaumaßnahmen des Gerätes vorgestellt. Mit dem Aufbau des Lasermassenspektrometers, das aus einem modifizierten Gaschromatograph/ Massenspektrometer mit einer Quadrupol-Ionenfalle besteht, steht ein Messverfahren zur Verfügung, dass eine kontinuierliche online-Messung mit kurzer Analysezeit, entfallender Probenvorbereitung, hoher Genauigkeit und Empfindlichkeit sowie einfacher Kalibrierung erlaubt. Um den Laserstrahl in das Massenspektrometer einzukoppeln, sind mechanische und elektronische Veränderungen am Gerät notwendig. Das Vakuumgehäuse des Massenspektrometers mit eingesetzter Quadrupol-Ionenfalle mussten für den Strahlengang des Ionisationslasers modifiziert werden. Das zu entwickelnde Gasanalysesystem nutzt alternierend die Elektronenstoß-Ionisation mit Filamentelektronen sowie die Laserionisation mit Photonen des Laserlichts (266 nm). Die quasi-kontinuierliche Aufnahme der Spektren erlaubt die Verfolgung des Musters der aromatischen Verbindungen in Echtzeit und die Optimierung des Vergasungsprozesses auf Basis der Messergebnisse. Bei kontinuierlicher Prozessüberwachung wird es möglich, den Teergehalt im Gas über die gesamte Zeit zu überwachen und auftretende Teerspitzen sofort zu erfassen. Der Vergasungsprozess kann dementsprechend angepasst werden.

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Poster P108 Aufreinigung und Analyse von Phosphopeptiden und His-Tag-Proteinen mittels eines funktionalisierten MALDI-Probentellers Tri Hoang 1, Ali Tinazli 2, Robert Tampé 2, Michael Karas 1 1

Institut für Pharmazeutische Chemie, Johann-Wolfgang-Goethe Universität Frankfurt a.M., 2 Institut für Biochemie, Johann-Wolfgang-Goethe Universität Frankfurt a.M. [email protected]

Phosphorylierung stellt eine der wichtigsten posttranslationalen Modifikationen von Proteinen dar. Reversible Phosphorylierung ist maßgeblich an der Regulation der Genexpression, des Zellwachstums und der Zelldifferenzierung beteiligt. Die Phosphorylierung eines Proteins kann dessen Eigenschaften beeinflussen, so z.B. dessen intrinsische biologische Aktivität. Zur Identifizierung der Phosphorylierungsstellen von Proteinen kommen vornehmlich ESIund MALDI-Techniken zum Einsatz. In der Regel wird das zu identifizierende Protein proteolytisch in Fragmente geschnitten, und die resultierenden Peptide anschließend massenspektrometrisch analysiert. Allerdings stellen Phosphopeptide in solchen Peptidgemischen sowohl für ESI als auch MALDI eine analytische Herausforderung dar. Die phosphorylierten Peptide sind stöchiometrisch unterrepräsentiert und die Ionisation der Phosphopeptide wird durch die Anwesenheit nicht-phosphorylierter Peptide supprimiert. Gängige Methode ist deshalb die Separierung der Phosphopeptide von den nicht phosphorylierten Peptiden vor der massenspektrometrischen Analyse mittels IMAC-Beads (Immobilized Metal Ion Affinity Chromatography). Hier wird eine Alternative zu der Bead-Technologie vorgestellt. Zum Einsatz kommt eine chemisch modifizierte MALDI-Telleroberfläche, die es ermöglicht, Phosphopeptide aus 1-2 µL eines komplexen Peptidgemisches zu isolieren und anschließend per MS zu analysieren. Vorteil der neuen Methode sind geringes Probenvolumen, hohe Sensitivität, schnelle Durchführung und minimierte Probenverluste. Da die hier vorgestelle Technik auf IMAC-Prinzipien basiert, ist neben der Aufreinigung von Phosphopeptiden auch die Anreicherung von intakten His-Tag-Proteinen aus komplexen Proben ein weiteres Anwendungsgebiet dieses modifizierten MALDIProbentellers. Anhand von Modellproteinen wird eine sensitive und schnelle Methode vorgestellt, mit der die Analyse von Phosphopeptiden und His-Tag-Proteinen wesentlich vereinfacht wird.

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Poster P109 LC-MS/MS and MS-BLAST for Automated Homology-Based Proteomics in Dunaliella salina Waridel P. 1, Katz A. 2, Pick U. 2 and Shevchenko A. 1 1

Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany. 2 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

Despite increasing progress in genomic sequencing, protein identification in many plants rely on homology-based proteomics. An essential tool for this strategy is MS BLAST, a sequence-similarity searching program, which uses redundant, degenerate and partially inaccurate sequences obtained from MS/MS spectra of peptides after tryptic digestion of proteins. With LC-MS/MS analyses are performed automatically, with a high speed, sensitivity and reproductibility. We investigated the performance of LC-MS/MS in combination with MS BLAST, using a linear trap instrument (LTQ). We tested the possibilities of automated de novo sequencing using the multiple stage MS and zoom scan capabilities of LTQ. We applied this technology for proteomic investigation of Dunaliella salina. This is a halotolerant green alga used as a model photosynthetic organism to investigate cell adaptation to saline stress. Differential plasma membrane protein expression of algal cells between moderate and high saline environment were studied to elucidate the cellular mechanisms of halophilic adaptation. LC-MS/MS was particularly adapted to the analysis of plasma membrane proteins, as we could only resolve them on 1D SDS-PAGE.

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Poster P110 Parallel PSD analysis of 2D-gel spots from E.coli M. Snel1, E. Claude1, D. Kenny1, T. McKenna1, Marc Kipping2 and J. Langridge1 1

Waters Corporation, MS Technologies Centre, Atlas Park, Simonsway, Manchester M22 5PP, UK. 2 Waters GmbH, Hauptstr. 85, 65760 Eschborn, Germany.

Peptide mass fingerprinting (PMF) is an established technique for identifying proteins. In PMF experiments, proteins are digested using a site selective protease, e.g. trypsin and the masses of the peptides produced are measured, typically using MALDI TOF mass spectrometery. Proteins are then identified by comparing these masses with those expected from an in silico digestion of proteins in protein sequence databanks. This approach can be very successful, but crucially relies on several factors: Proteins under investigation must be present in the databank being interrogated. Proteins must be isolated effectively from other proteins, as protein mixtures lead to ambiguous databank search results. Proteolytic digestion must produce at least four peptides falling in the m/z range of 600 to 4000 and yielding mass spectral peaks of similar intensity. Due to these limitations, between 10% - 60% of proteins studied are not identified using PMF. The specificity of protein identification experiments can be enhanced, by obtaining partial sequence information from these peptides by MS/MS or PSD experiments. Here we present an evaluation of a novel post source decay (PSD) experiment as an enhancement to PMF. The PSD experiment described differs from conventional PSD, as data are obtained simultaneously from a number of peptide ions. Parallel acquisition is made possible by assigning PSD fragments to associated precursor ions by determining the kinetic energy of PSD fragments. This novel approach simplifies the PSD experiment, reduces sample consumption and increases the number of peptides analysed. A comparison of parallel PSD and PMF was made using a 2d gel separated cytosolic fraction of E. coli. Improvement of the rate of protein identification is demonstrated.

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Poster P111 A novel strategy for the analysis of phosphopeptides, coupling N-terminal peptide derivatisation and HPLC separation with mass spectrometry M. Snel1, I. Campuzano1, E. Claude1, T. McKenna1, Marc Kipping2 and J. Langridge1 1

Waters Corporation, MS Technologies Centre, Atlas Park, Simonsway, Manchester M22 5PP, UK. 2Waters GmbH, Hauptstr. 85, 65760 Eschborn, Germany.

Protein phosphorylation is the most common post translational modification that occurs in mammalian systems and is especially important in a number of key biological processes, such as intracellular signalling and facilitates the method of action of several key hormones. However, the analysis of small hydrophilic phosphopeptides by LC-MS and LC-MS/MS techniques is particularly challenging due to their poor retention characteristics on reverse phase media. During the HPLC experiment they are often ‘lost’, either by not binding to the trapping column during the loading step often employed in nano LC or by eluting in the void volume for a direct loading LC experiment. The analysis of these species by either ESI or MALDI MS is complicated by their low molecular weight. Here we describe the N-terminal derivatization of small phosphopeptides using N-Tris (2,4,6-trimethoxyphenyl) phosphonium-acetic acid N-hydroxysuccinimide ester (TMPPacOSu). This improves the phosphopeptide retention on reverse phase material and increases the mass of the peptide by 572 Da. We will present data showing improved chromatographic performance of small hydrophilic peptides after derivatisations, showing increased retention on C18 reverse phase columns as well as effective binding to trapping columns. MS data were acquired on-line, suing an ESI TOF instrument and off-line using a MALDI Q-Tof instrument.

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Poster P112 NanoLC/MS In Quantitative and Qualitative Proteomics Jeff. W. Finch1, Hong Ji Liu1, Jim Langridge2, Geoff Gerhardt1, Matthew Kennedy3, Steve. A. Cohen1 & K. Fadgen1 1

Waters Corporation, 34 Maple Street, Milford Massachusetts, 01757 USA. 2Waters Corporation, MS Technologies Centre, Atlas Park, Simonsway, Manchester M22 5PP, UK. 3 Waters Corporation, Transistor Straat 18, 1322 CE Almere, Netherlands. Among the improvements in proteomics analyses is use of nanoscale liquid chromatography (nanoLC) where the increase in MS sensitivity obtained is critical when sample-limited. Here we describe a design for nanoLC/MS that overcomes reproducibility problems associated with traditional split flow solvent delivery systems. Specified flow rates in the range of 200 – 5000 nl/min are compatible with 75 -320 um ID columns, and by avoiding stream splitting, provides reliable, reproducible results. Separations of complex cellular extracts or biofluids can be accomplished with component retention times varying less than 10 seconds over extended sample sets. Examples with gradient times ranging from 30 – 120 minutes will be shown. This performance is critical for comparing samples quantitatively, and ensuring that differences amongst samples and controls are not an artifact of the separation step. System components are designed for new small particle columns requiring control of system bandspread to limit peak broadening. Comparisons of complex mixture analysis performed on 3.0 and 1.7 um particle columns shows that separations with the smaller particle columns yield peak widths half of the large particle ones. The enhanced peak capacity attained is beneficial for analysis of complex sample mixtures, simplifying the interpretation of MS and MS/MS data, and the reduced peak volumes also result in significantly greater peak response, routinely achieving low femtomole to attomole sensitivity on 75 um ID columns. Both quantitative and qualitative comparisons of complex extract digests show clear advantages through increased resolution and reproducibility that are essential for biomarker identification studies.

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Poster P113 A novel LC-MS based approach to qualitative and quantitative protein profiling and biomarker discovery Scott Geromanos1, Keith Richardson2, Phillip Young2, Richard Denny2, Kieran Neeson2, Therese McKenna2, Matthew Kennedy3, Craig Doreschel1, Marc Gorenstein1, Guo-Zhong Li1, Timothy Riley1, Jeffrey C. Silva1 and James Langridge2 1

Waters Corporation, 34 Maple Street, Milford Massachusetts, 01757 USA. 2Waters Corporation, MS Technologies Centre, Atlas Park, Simonsway, Manchester M22 5PP, UK. 3 Waters Corporation, Transistor Straat 18, 1322 CE Almere, Netherlands. Current methodologies for protein quantitation include 2DE Gel Electrophoresis, stable isotope coded affinity tags (ICAT, GIST), metabolic labeling (15N), and stable isotope labeling with amino acids in cell culture (SILAC) to name only a few. Perusal of the current literature clearly illustrates both pros and cons for each of the previously mentioned methodologies. In this presentation we will present a new concept for qualitative and quantitiative protein profiling. This is based upon a simple LC-MS based methodology that will allow for determining the relative change in abundance of proteins in highly complex mixtures. Utilizing a reproducible chromatographic separations system along with the high mass resolution and mass accuracy of an orthogonal time-of-flight mass spectrometer, allows for the identification of tens of thousands of ions emanating from identically prepared control and experimental samples. Using this configuration, we can determine the change in relative abundance of a small number of ions between the two conditions solely by accurate mass and retention time. Our data clearly shows that with respect to digestion, ionization, and chromatographic reproducibility as well as high mass precision we are capable of generating the appropriate levels of reproducibility and mass precision to provide such conclusions and in addition that the associated algorithms are capable of extracting the data and calculating the appropriate responses with careful attention to proper error modelling. In addition this technology provides a powerful MS based peptide identification alternative to conventional LC-MS/MS strategies. In the course of an LC-MS acquisition the collision energy is continuously switching from low to high energy throughout the entire LCMS acquisition. The resulting high-energy data provides extensive fragmentation information across the entire mass range, including the low mass region, across the entire peak width for every precursor ion detected in the low energy function of the chromatogram. The fragment ions (high energy function) are aligned to their related precursor ions in chromatographic space by retention time and chromatographic peak shape. The results clearly demonstrate the ability to confidently identify significantly more proteins with higher sequence coverage than traditional DDA based approaches as well as minor peptide components within a complex peptide mixture. Results from the study of various biological samples such as E.coli and Human plasma and serum will be presented.

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Poster P114 High Performance Proteasome Proteomics using UV- and IR-MALDI FTICR Mass Spectrometry Reinhold Weber1, Nikolay Youhnovski1, Regina Preywisch2, Marcus Groettrup2 and Michael Przybylski1 1

Laboratory of Analytical Chemistry, Department of Chemistry, University of Konstanz, 78467 Konstanz, Germany. 2 Laboratory of Immunology, Department of Biology, University of Konstanz, 78467 Konstanz, Germany

For several years ubiquitin-mediated intracellular protein degradation is a focus area of biochemical research [1]. A protein to be degraded (e.g. due to misfolding or translation errors) is labelled with ubiquitin by the action of enzymes E1 – E3. The marked protein is degraded by the proteasome, ubiquitin is recycled, and the remaining peptides serve as ligands for MHC class I molecules to be presented to CTL. The proteasome consists of two parts: The cylindrical shaped, proteolytically active 20S proteasome is capped by regulatory 19S proteasomes. The 20S proteasome consists of two rings of each seven different - und -subunits. Upon an infection, cytokines such as interferon  are released, leading to a reorganisation of the molecular composition of the 20S proteasome: Inducible -subunits replace the constitutive subunits [2]. The proteomics approach presented here for the molecular mapping of 20S proteasomes includes proteasome isolation from different tissues (mouse intestine and liver), 2 D gel electrophoretic separation of the single subunits, staining and tryptic in-gel digestion. The subsequent high resolution FT-ICR mass spectrometric analysis has been carried out using both UV- and IR-MALDI. A major advantage of IR-MALDI is the “softer” ionisation in comparison to standard UV-MALDI, avoiding photochemical fragmentation of labile peptides and post-translational modifications [3]. The high accuracy of mass determination allows direct assignment of sequences to the corresponding peptides and unambiguous identification of the proteasome subunits. The influence of laser irradiation on ion formation is pointed out by a comparison of the abundances of pyro-glutamic acid and glutamic acid using UV- and IR-MALDI. Literatur [1] A. Hershko, A. Ciechanover, A. Varshavsky, Nature Med. 6, 10 (2000) [2] K. Schwarz, M. van den Broek, S. Kostka, R. Kraft, A. Soza, G. Schmidtke, P.-M. Kloetzl, M. Groettrup, J. Immunol. 165, 768 (2000) [3] B. A. Budnik, K. B. Jensen, T. J. D. Jǿrgensen, A. Haase, R. A. Zubarev, Rapid Commun. Mass Spectrom. 14, 578 (2000)

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Poster P115 Mass Spectrometric Proteome Analysis of Skeletal Muscle Tissue from Inbred Fat Mouse Strain in Comparison to Normal Weight Mouse Lines; On the Way to Detect Marker Proteins for Obesity M.R Wiechert1, G.A Brockmann2, U. Renne3, M.O.Glocker1 1

Proteome Center Rostock, University of Rostock, Rostock, Germany, 2Institute for Animals Sciences, Humbold-Universität zu Berlin, Berlin, Germany, 3Research Institute for the Biology of Farm Animals, Dummerstorf, Germany Clinical background: The number of obese people is increasing continuously, associated with a multitude of medical, psychological and economic problems. In Germany almost 40% of the population and 25% ofall children are overweight. Obesity is influenced by genetic factors, so that individual behaviour cannot be regarded as its sole cause. Hence, it is necessary to understand the aetiology and the molecular mechanisms to search systematically for prevention- and treatment possibilities. Skeletal muscle has high effects on general metabolism and is the principal site of insulin-stimulated glucose uptake. Due to the muscle mass, normal and pathological fluctuations of the level of several hormones (insulin, growth hormone, catecholamines, glucocorticoids and interleukin) lead to changes of turnover in the muscle cells and as a result in the whole body. Thus, studying protein expression differences in muscle tissue may lead to marker proteins for obesity. Research project: With the aim to find differentially expressed proteins, we studied the line DU6i, which is more than two times as heavy and three times as fat as nonselected controls. Animals are suffering from hyperleptinemia, hyperinsulinemia and significantly elevated serum IGF-I concentrations at the age of above six weeks. The genetically related but randomly mated mouse line DUKsi was chosen as control together with the commercial line DBA/2 [1]. Skeletal muscle tissue samples are taken from 6-week-old individuals and subjected to a global proteome research approach [2]. Coomassie-blue stained twodimensional gels of protein extracts from tissue samples of three to four animals per line were individually compared by computer-assisted image analysis. Each gel contained about 1000 spots. Overall appearance of the spot pattern is well comparable between the investigated groups showing the high reproducibility of the technique. Close inspection of the gel images revealed that seven protein spots were different in abundance between the DU6i and the DUKsi lines. The same spot differences were observed in the comparison of DU6i to DBA/2 lines, whereas the comparison of the DUKsi lines with the DBA/2 mice showed no differences. Subsequently, spots are analysed by matrix-assisted laser desorption/ionisation time of flight mass spectrometric peptide mass fingerprinting in order to identify the differentially expressed proteins. Further investigations shall evaluate the applicability of the identified proteins as obesity markers. References [1] G.A Brockmann. and M.R Bevova. (2002). TRENDS in Genetics 18, 467-375. [2] A. Sinz., M. Bantscheff, S. Mikkat, B. Ringel, S. Drynda, J. Kekow, H.-J. Thiesen, and M. O. Glocker (2002). Electrophoresis, 23, 3445-3456.

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Poster P116 A novel approach to stable isotope protein labeling for quantitative proteomics Sebastian Wiese1, Kai Reidegeld1 und Bettina Warscheid1 1

Medical Proteom-Center, Ruhr-Universität Bochum

Quantitative proteome analysis represents a powerful tool to gain deeper insight into biological systems. Besides the differential display of soluble proteins via two-dimensional gel electrophoresis (2-D PAGE), stable isotope labeling in combination with mass spectrometry (MS) is a suitable method to obtain relative quantitative information on proteins in complex biological samples. In contrast to metabolic labeling experiments in which the gene products are isotopically modified, most chemical labeling approaches are only applicable to the proteolytic peptide fragments of the extracted proteins, which may result in less accurate protein quantitation. A general advantage of protein labeling prior enzymatic digestion is reflected in the possibility to reduce the complexity of biological samples by means of protein separation techniques (e.g., gel electrophoresis). In this work, we describe a chemical approach to modify intact proteins with isotope-coded tags for quantitative MS analysis. For relative quantitation, two protein samples are labeled with two isotopically different tags and subsequently combined for separation by 1-D or 2-D PAGE. Proteins are excised and treated with protease for in-gel digestion. The proteolytic peptide mixtures generated were further analyzed by nano high-performance liquid chromatography (nanoHPLC) coupled to electrospray ionization (ESI) MS. Mass spectral data evaluation for the determination of protein concentration ratios was performed with the in-house developed software package Peakardt. The isotope-coded labeling reagent applied is shown to specifically react with the -amino group of lysine as well as the free N-terminal amino group of proteins. Mass spectral data indicates the completeness of the labeling reaction and a good linearity of relative quantitation was obtained with a standard protein mixture prepared in six different concentration ratios. Additionally, the applicability of the reported labeling method to protein separation by 1- and 2-D PAGE is displayed, and fragmentation pattern of tagged versus unmodified peptides are investigated.

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Poster P117 ESI-MS-Untersuchungen zur intrazellulären Phosphorylierung des Drosophila Transmembranproteins Crumbs Nicola Wiethölter1, Susann Fornaçon2, Elisabeth Knust2, Sabine Metzger1 1

BMFZ-Analytisches Zentrallabor, Uniklinik Düsseldorf, Moorenstraße 5, 40225 Düsseldorf 2 Institut für Genetik, HHU Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf

In Drosophila melanogaster ist der Crumbs Komplex für die Ausbildung und Aufrechterhaltung der Zonula adherens essentiell, einer Zellstruktur, die die enge Adhäsion von Epithelzellen untereinander gewährleistet. Bei dem Crumbs Protein handelt es sich um ein Transmembranprotein mit einer großen extrazellulären Domäne und einer kleinen nur 37 Aminosäuren langen intrazellulären Domäne. Letztere ist essentiell für die Funktion und es ist bekannt, dass sie Bestandteil eines Multiproteinkomplexes ist. Durch Co-Immunopräzipitationsexperimente mit anschließenden ESI-MS/MS Analysen wurde nach neuen Bindungspartnern der intrazellulären Domäne gesucht und diese Domäne näher charakterisiert. Es konnte nachgewiesen werden, dass ein Peptid der intrazellulären Domäne des Crumbs-Proteins phosphoryliert ist. Das phosporylierte Crumbs-Peptid ist 14 Aminosäuren lang und hat 5 (1 Threonin, 2 Serin, 2 Tyrosin) potentielle Phoshorylierungstellen. Die Ergebnisse der Untersuchungen am nativen phosphorylierten Crumbs-Peptid und an synthetisch hergestellten phosphorylierten Crumbs-Peptiden sollen Diskussionspunkte dieses Beitrags sein.

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Poster P118 Capillary Electrophoresis-ESI-FTMS: Accurate Mass Measurement of Amino Acids in Lysated Bacterial Cells Matthias Witt 1, Christian Neusüss 2, Edward Baidoo 3 1

Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen. 2 Bruker Daltonik GmbH, Permoserstr. 15, 04318 Leipzig. 3 Department of Chemical Engineering, University of California at Berkeley, CA 94720, USA.

Capillary electrophoresis-mass spectrometry (CE-MS) is a well accepted multidimentional analytical approach complementary to the LC/MS technique [1]. Fourier Transform Mass Spectrometry (FTMS) is known as a technique for very accurate mass measurements. Due to the complexity of bacterial lysates which requires a mass spectrometer with high resolution and accurate mass this technique has been coupled to a CE system for the separation of polar compounds like amino acids using electrospray ionization with a special CE interface. The measurements have been performed with the APEX Qe 7T system (Bruker Daltonik GmbH, Bremen, Germany) equipped with the Apollo electrospray source and a Hewlett Packard 3DCE (Agilent, Waldborn, Germany) system. In this setup a coaxial sheath-liquidinterface has been used for high robustness. This interface based on a grounded ESI needle provides sensitive microspray ionization and stable spray conditions [2]. Amino acid concentrations down to low M concentrations have been detected by CEFTMS with mass accuracies in the low ppm range. All amino acids have been separated by CE in less than 12 minutes with a typical peaks width of about 6-10 seconds. Different amounts of an amino acid standard have been measured for the verification of quantitative results. The high potential of combining the high resolution and accurate mass of FTMS with the fast separation and high efficiency of capillary electrophoresis is demonstrated.

Literatur: [1] P. Schmitt-Kopplin, M. Frommberger, Electrophoresis 2003, 24, 3837-3867. [2] C. Neusüß, M. Pelzing, M. Macht, Electrophoresis 2002, 23, 3149-3159.

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Poster P119 Neue Strategien in der Strukturaufklärung durch LC/FT-MS Rainer Wolf, Michael Simon, BASF Aktiengesellschaft, Kompetenzzentrum Analytik, Spektroskopie GKA/S 67056 Ludwigshafen, Germany Die Strukturaufklärung in Verbindung mit der LC/MS basiert in der Regel auf der Bestimmung des Molekulargewichts der zu untersuchenden Verbindung sowie den dazugehörigen MS/MS-Spektren. Der Einsatz von Datenbanken spielt in der LC/MS nur eine untergeordnete Rolle, es gibt keine größeren kommerziell erhältlichen Spektrensammlungen. Mit der Verfügbarkeit der Time off Flight - Massenspektrometer (TOF-MS) können auch in der Routine-LC/MS akkurate Massen mit einer Präzision < 5 ppm bestimmt werden. Aus diesen akkuraten Massen können Summenformeln abgeleitet werden, die neben den MS/MS -Spektren eine der wichtigsten Informationen für die Strukturaufklärung darstellen. Während MS/MS-Spektren vor allem dann von Bedeutung sind, wenn es um eine abgegrenzte Substanzklasse oder wiederkehrende Problemstellungen geht, liegt der Nutzen der Summenformel vor allem bei der Bedeutung neuer oder einmaliger Fragestellungen. Die wesentliche Einschränkung bei der Bestimmung von Summenformeln unbekannter Verbindungen liegt in der großen Anzahl möglicher Element-Zusammensetzungen, wenn man nur wenig Einschränkungen bei den zur Berechnung zugelassenen Elementen macht. Die Anzahl der Element-Zusammensetzungen wird um so kleiner, je präziser die gemessene Masse oder je eingeschränkter die Anzahl zugelassener Elemente ist. Für eine schnelle Routineanalytik sollten diese Einschränkungen so klein wie möglich sein. Eine weitere Information für die Strukturaufklärung liegt in den Isotopenmustern der gemessenen Molekülionen. Einige aktuelle Softwarepakete enthalten bereits Tools, die auf der Basis der Nominalmassenauflösung die Höhe der Isotopen verwenden, um die Anzahl möglicher Summenformeln zu verringern. Deutlich mehr Informationen erhält man durch Einsatz der Fourier-TransformMassenspektrometrie (FT-MS) in der Strukturaufklärung. Einerseits lassen sich mit dieser Technik Massen mit einer Präzision < 0,5 ppm zu bestimmen. Andererseits ist man in der Lage, Isotopenmuster so hoch aufzulösen, dass man mit der Hilfe der Feinstruktur Aussagen über vorliegende Elemente machen kann. Mit diesen beiden Informationen (akkurate Masse und hochaufgelöstes Isotopenmuster) lassen sich neue Strategien für die Strukturaufklärung entwickeln. Mit der genauen Kenntnis der akkuraten Masse ist es z.B. sinnvoll, nicht nur in Spektren-, sondern auch in Strukturdatenbanken zu recherchieren. Das gemessene hochaufgelöste Isotopenmuster wird mit einem simulierten verglichen und damit die Anzahl möglicher Summenformeln deutlich reduziert

226

Poster P120 Determination of ganglioside expression in human gliosarcoma by chip electrospray tandem mass spectrometry Alina D. Zamfir, Laura Bindila, Željka Vukelić, Jasna Peter-Katalinić Institute for Medical Physics and Biophysics, University of Münster Gliosarcoma (GSa) is a rare primary neoplasm of the central nervous system classified as a variant of glioblastoma, therefore a grade IV tumor [1]. One of the factors leading to therapeutic failure in the treatment of human GSa [2] is that the tumor cells exhibit extensive infiltration into the surrounding normal brain tissue and therefore they are inaccessible to current treatment methods. One strategy to target invading tumor cells is to use ligands that bind specifically the cells, however the critical point in such a strategy is to identify the tumor-specific targets for such ligands. One group of potential molecular targets are gangliosides, which are frequently differentially expressed in gliomas [3]. Moreover, their expression is reflected also in the malignancy grade of the tumor. For these reasons the determination of ganglioside expression in human glioma tumor is of major biological, clinical and therapeutic importance. Here we report upon the mapping and structural investigation of gangliosides extracted from human gliosarcoma by fully automated chip electrospray (ESI) quadrupole time-of-flight (QTOF) tandem mass spectrometry. The high sensitivity and ionization efficiency provided at nanoscale level by the chip ESI MS [4,5] made this advanced technology ideal for such applications that require identification of unknown, minor components in complex ganglioside mixtures. The optimized chip ESI QTOF tandem MS method was capable to ionize, detect and sequence a significant number of ganglioside components specific for this type of malignant tissue transformation. Additionally, the stable spray provided by the chip ESI infusion allowed fragmentation analysis and detailed structural elucidation of different minor ganglioside structures among which, variants of GM3, previously suggested [3] as a chemotherapeutic agent for human high-grade gliomas and the low abundant O-Ac GD3 species presently investigated as potential biologically relevant component in the tumor cells. Our study documents here for the first time the position of the O-Acetyl group along the saccharide chain, which may contribute to a better understanding of the biological functionality of this species.

References [1] Alatakis, S., Stuckey, S., Siu, K., McLean, C. J Clin Neurosci. 2004, 11, 650-656 [2] Grossman, S.A., Batara, J.F. Semin. Oncol. 2004, 31, 635-644 [3] Noll, E.N., Lin, J., Nakatsuji, Y., Miller, R.H., Black, P.M., Exp Neurol. 2001, 168, 300-309 [4] Zamfir, A., Vukelić, Z., Bindila, L., Peter-Katalinić, J., Almeida, R., Sterling, A., Allen, M. J. Am. Soc. Mass Spectrom. 2004, 15,1649-1657 [5] Zamfir, A., Lion, N., Vukelić, Ž., Bindila, L., Rossier, J., Girault, H. H., Peter-Katalinić, J. Lab Chip 2005 (in press).

227

Poster P121 De Novo Sequencing using MALDI QIT TOF MSn – Application of a Novel Sequencing Algorithm to Clinical Proteome Samples H. Montgomery 1), C. Koy 2), M. May 3), J. Broughton 3), M. Resch 4) S. Iwamoto 5), M.O. Glocker 2), and K. Tanaka 5) 5) Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu, UK 6) Proteome Center Rostock, University of Rostock, Rostock, Germany. 7) Shimadzu Research Laboratory Ltd., Manchester, UK. 8) Shimadzu Germany, Duisburg, Germany. 9) Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu, Japan. In proteome research the importance of de novo sequencing is increasing as it offers new possibilities in the analysis of protein samples from organisms that are not yet completely sequenced and in order to pin-point post-translational modifications. Also, in cases where proteins are not completely separated in 2D gel spots or 1D gel bands, sequencing of peptide ions enables the reliable identification of the overlaying proteins. In an attempt to identify human plasma proteins of lower abundance, samples from patients suffering from Rheumatoid Arthritis [1] were fractionated by continuous preparative freeflow electrophoresis [2, 3], followed by HPLC and 1D SDS PAGE. Protein bands of interest were excised, tryptically digested and analyzed by MALDI QIT ToF MSn. Acquired MSn data were subjected to de novo sequencing using Shimadzu Research Laboratory’s proprietary software and other commercially available packages. Using this approach, clear identification of up to three overlaying proteins per band with more than one sequenced peptide was achieved. Some of the identified proteins such as antithrombin III, actin beta, and pancreatic ribonuclease do not belong to the group of abundant plasma proteins, and are rather addressed as so called “leakage proteins”. An intriguing observation is that although the human genome is supposed to be completely sequenced, some proteins found in plasma cannot be identified as being from human origin using the sequence information. Instead, sequence data suggest the presence of other, related protein sequences originating from other organisms. This may be taken as an indication that hitherto unknown sequence variations of a particular protein are present in the patient samples and can be detected using the outlined approach. Our results show, that mass spectrometric sequencing is clearly helping with in-depth analysis of complex proteome samples and opens the way for protein structure analysis. Literature [1] A. Sinz, M. Bantscheff, S. Mikkat, B. Ringel, S. Drynda, J. Kekow, H.-J. Thiesen, and M. O. Glocker, Electrophoresis, 2002, 23, 3445-3456. [2] B. Ringel, S. Drynda, I. Schinke, S. Mikkat, H.-J. Thiesen, G. J. Eppert, J. Kekow, and M. O. Glocker, Proc. Swiss Proteomics Soc., 2003, 56-57. [3] M.O. Glocker, C. Koy, P. Serrano-Fernandez, M. Kreutzer, S. Möller, B. Ringel, S. Mikkat, R. Martin, S. Drynda, J. Kekow, and H.-J. Thiesen, Conf. Proc. 6th. Siena Meeting, Siena, 2004.

228

Poster P122 Synthesis and structural characterization of polyubiquitin conjugates using high resolution mass spectrometry Ji Eun, Jung1* , Marilena Manea2, Martin Scheffner,2 Michael Przybylski1 1

Laboratory of Analytical Chemistry, Department of Chemistry, University of Konstanz, 78457 Konstanz Germany 2 Laboratory of Biochemistry, Department of Biology, University of Konstanz, 78457 Konstanz Germany

Modification of proteins by covalent attachment of ubiquitin plays a fundamental role in the control of many biological processes including cell cycle regulation, transcription, DNA repair, and apoptosis (1, 2). Ubiquitin contains 7 lysine residues each of which in principle could be used for polyubiquitin conjugations and chain formation. Importantly, recent evidence indicates that the actual lysine residue of ubiquitin used for ubiquitin-ubiquitin conjugation determines the biochemical/biological function of the respective polyubiquitin chain. Therefore, the synthesis and structural characterization of a series of chemically defined polyubiquitin chains that differ by the lysine residue used for ubiquitin-ubiquitin attachement is a major goal of the present work. In order to synthesize such conjugates specific chemical strategies for the build-up of polyubiquitin chains are pursued that use thioether ligation of C-terminal ubiquitin-thiol residues with N-(chloroacetyl)-side chain protected Lysine residues and contain specific and original lysine residues at each distinct branching site (3). High resolution FT-ICR mass spectrometry (FTICR-MS) was predominantly used for the characterization of the chemically synthesized polyubiquitin chains, and affinity proteomics methods are especially suitable for identifying the reconjugation structures of polyubiquitin.

[1] Nobel Prize in Chemistry, 2004 awarded to Aaron Ciechanover, Avram Hershko, and Irwin Rose for the discovery of ubiquitin-mediated protein degradation; Press release, Swedish Academy of Sciences. [2] L.K. Linares , A Hengstermann , A Ciechanover , S Müller and M Scheffner (2003) Proc, Natl. Acad. Sci USA. 100: 12009 - 12014. [3] G. Mezö, M. Manea, A. Jakob, B. Kapuvari, S. Bösze, G. Schlosser, M. Przybylski, F. Hudecz (2004) J. Peptide Sci, 10, 701-713.

229

Autorenverzeichnis / Author Index

Abbass, Karen Aebersold, Ruedi Affolter, M. Ahlmann, Norman Ahrends, R . Allen, Mark Alpert, C. Altug, Zekeriya Anisimov, A. P. Argenzio, Elisabetta Asperger, Arndt Bader, S. Baidoo, Edward Bandur, R. Barth, Hans-Dieter Bartoszek, M. Bataineh, M. Bateman, Bob Bauer, Andreas Baumann, Christian Baumann, S. Baumbach, J. I. Baykut, Gökhan Becker, J. S. Becker, J. Sabine Becker, J. Susanne Beck-Sickinger, Annette G. Behrendt, Frank Beitz, T. Bellahn, I. Bergmann, Ulf Bergonzelli, G. E. Berkenkamp, Stefan Bernhard, Marco Besa, Axel Besche, S. Bessenyei, S. Bildl, Wolfgang Bindila, Laura Biniossek, Martin Birch, James M. Blaut, M. Bleiholder, Christian Blüggel, Martin Boesl, Ulrich Bogenschütz, German Bogumil, R. Borchert, H. H. Bornschlegl, Alexander Borsotti, Dario Bouschen, W. Brand, Sven Brandt, R.

V113 V074 V071 P43 P1 V041 P22 P32, V081 V045 V082 P73 P4 P118 P45, V034 V021 P2 P74 V022 P8 V075 V063, V073 P3, P4, P5, V034, V061 V041 P10, P11, P9 P6, P8, V011 P6, P7, V011 P91 P107 P59 P12 P13 V071 V052 V032 V091 V034 P5 V113 P120, P14, V041 V113 V095 P22 P15 V015 V035 P78 P68 P42 V035 V082 P16, P41, P64 P36 P45

Brennen, Reid Brenner, Christian Brockmann, G. A. Brose, Nils Broughton, J. Brown, Jeff Brutschy, Bernhard Buback, M. Bucci, Nadine Bukau, Bernd Bulau, Patrick Burhenne, Heike Campuzano, I. Carling, D. Ceglarek, U. Chakraborty, P. Chamrad, Daniel C. Chen, H. Christ, P. Christoph, H. Ciosto, Cornelia Cittaro, Davide Claude, E. Coelho, F. Coffee, Keith R. Cohen, Steve. A. Consorti, C. S. Cooper, Dermot M. F. Corvey, Carsten Corvini, P. Czerwieniec, Gregg A. Damoc, E. Danikiewicz, Witold Dankbar, Nico Dawes, E. Dawes, P. Dehnhardt, Markus Deininger, Sören-Oliver Deller, Thomas Demelbauer, Uwe Denny, Richard Diederichsen, U. Diezemann, N. Dijkwel, P. P. Dolatowska, K. Doreschel, Craig Dreisewerd, Klaus Dresen, S. Drewelow, B. Drhlkkovi, Tatina Drynda, S. Dumitrel, A. Duncan, Wayne P. Dupont, J. Duursma, Mark Eberlin, M. N. Eble, Silke Eckart, Klaus Edelson-Averbukh, Marina

V054 P82 P115 V112 P121 V022 V021 P25 P77 P33 P17 P53 P111 P12 V063, V073 P24 V015 P45 P18 P19 P90 V082 P110, P111 V124 V095 P112 V124 P91 P67 V065 V095 P70 HV4, P104 V053 P23 P23 P8, V011 P73 V113 P21, P34, V042 P113 P24 P24 P37 P104 P113 V052 V092 P89 P87 P54, P84, V072 P7 P26 V124 P85 V124 V113 V112 P60

Edler, Michael Eggers, Uwe Eickelberg, Oliver Ejsing, C. S. Elend, Manfred Engst, W. Eppert, G. J. Erhard, M. Eubeler, Jan Fabbretti, Francesco Fadgen, K. Fakler, Bernd Falk, K. Farwanah, Hany Fay, L. B. Fergenson, David P. Fiedler, G. M. Fiehn, O. Finch, Jeff. W. Fischer, David Fischer, Steven M. Flock, Bianca Folkers, G. Fornaeon, Susann Frank, Matthias Franke, G. Franzke, Joachim Frauendorf, H. Freitag, L. Friedhoff, P. Friedrich, Alexander W. Froesch, Martin Fuchs, P. Fulda, Sabine Fürmeier, Sven Fürstenberg, S. Gaber, Angela Gafumbegete, Evariste Garcka-Villar, Natividad Gard, Eric E. Gedig, Erk Gerhardt, Geoff Geromanos, Scott Getie, Melkamu Geyer, Hildegard Geyer, Roland Geyer, Rudolf Girault, Hubert H. Glocker, M. O. Glückmann, Matthias Goethel, S. Gohlke, R. Gorenstein, Marc Graf, Cristian Gramberg, Jan Griep-Raming, Jens Groettrup, Marcus Gröger, T.

P43 P86 P17 V062 V121 P22 P84 P103 P80 P51 P112 V113 P68, V072 P82 V071 V095 V063, V073 V064 P112 P94 P26 P77 P70 P117 V095 V114 P43 P24, P25 P3, V061 P1 P65 V041 P89 P69 P28 P101 P32, V081 P49 V014 V095 V053 P112 P113 P29 P30 V033 P30 V041 P19, P38, P49, P54, P84, P115, P121, V072 P44 F02 P19, P38 P113 P33 P49 P31 P114 V064

Gross, Jürgen H. Grote, Jens Grote, M. Grotemeyer, Jürgen

P34, P77 V053 P74 P32, P57, P72, P83, P86, V081 Grundmann, A. P5, V034 Gühler, A. P22 Guillaume, E. V071 Gunzelmann, Karl-Heinz P35 Günzler, F. P25 Haehn, Sebastian P50 Hagemann, Martin P69 Hahner, Stefanie P36 Haid, Mark P55 Halicz, L. P9 Hamon, Christian V075 Hartig, Lutz P20 Hartmann, M. P5 Hau, J. V071 Hebeler, R. P37 Heeren, Ron P85 Heinzle, Elmar P106 Heisenberg, Carl-Philipp P62 Heitner, J. P38 Hepp, W. F08 Herr, Wolfgang P44 Herrmann, W. P42 Herschbach, H. P40 Hester, A. P41 Heumann, K. G. P45 Hille, R. P10 Hillenkamp, Franz V052 Hoang, Tri P108 Hochkirch, U. P42 Hoenicke, Katrin P20 Hoffmann, P. V102 Hoffmann, R. V014, V102 Hoffmann, T. P43, P45, P93, P98, V034 Hofmann, Sandra P44, P67 Holdt, H.-J. P101 Huenerbein, Andreas P88 Hung, Chien-Wen P60 Hunger, Corina P46 Hutmacher, Hans-Martin P35 Ihling, Christian P47, P50, P91, P95, V111 Ingendoh, Arnd V121 Iwamoto, S. P121 Iwersen-Bergmann, Stefanie V093 Izmer, A. P11, P9 Jabs, W. P36 Jackson, S. P12 Jahn, Olaf V112 Jakubowski, N. P45 Jaskolla, T. P48 Jebanathirajah, Judith A. V101 Jung J. P122 Junge, Harald V112 Just, Tino P49 Kaever, Volkhard P53

Kalkhof, Stefan Kallies, B. Kallow, W. Karas, Michael Karch, Helge Karst, Uwe Karstens, Michael Katz, A. Kauert, Gerold F. Kausche, Sandra Kayser, M. Kekow, J. Kellermann, Josef Kempf, J. Kennedy, Matthew Kenny, Daniel Keßler, Irina Kienle, Stefan Killeen, Kevin Kimmel, D. Kipping, Marc Kirsch, Dieter Kirschner, Marc W. Klein, Björn Kleinpeter, E. Klemm, Clementine Klöcker, Nikolaj Kluge, H.-Jürgen Knaack, Larisa Knaus, Hans-Günther Knepper, Thomas P. Knirel, YA. Knust, Elisabeth Koal, Therese Koch, Sonja Köhler, F. Kölbl, S. König, Simone Köstler, Martin Kostrzewa, Markus Koy, Cornelia Kramer, Achim Krause, Eberhard Krauß, Gudrun Kreutzer, M. Krüger, Ralf Kühlberg, Axel Kühn, Eva Kuhn, Karsten Kühne, Daniel Kuhnt, Christine Kulle, Alexandra E. Kunze, Kerstin Kussmann, M. Kuster, Bernhard Kutchan, Toni M. Lamer, Stefanie Lange, F. Langridge, J.

P50, P91, P95, V111 P59 P103 P44, P46, P48, P67, P108 P65 P61 P86 P109 V093 P44 P11 P54, P84, V072 P90 V092 P112, P113 P110, V022 P96 V075 V054 F06 P110, P111, V022 P1, P85, V051, V083, V094 V101 P51 P101 V013 V113 HV3 P52 V113 P78, P79, P80, V032 V045 P117 P53 P96 V094 P71, V044 V053 V051 P96 P38, P54, P84, P121, V072 V103 P105, V013 V033 P38 P60, V104 P55 P56 V075 V122 V125 P57 P43 V071 V074 V125 P105 P58 P110, P111, P112, P113

Langrock, Tobias Laudien, R. Lebrilla, Carlito Lehmann, Wolf D. Lehn, J. M. Leichtle, A. Leisner, A. Leize, E. Lembcke, J. Lenz, Christof Levsen, Karsten Li, Guo-Zhong Liesener, André Linden, H. Bernhard Lindner, B. Link, Vinzenz Linscheid, M.

V014 P59 V095 P60, V104 P40 V063, V073 P41, P64 P40 V063, V073 V024 V121 P113 P61 P31, F01 V045 P62 P13, P42, P66, P81, P97, P99, P100 Lion, Niels V041 Liu, Hong Ji P112 Lochnit, Günter P17 Loftus, N. F07 Lohaus, Christiane P63, P92 Löhmannsröben, H. -G. P59 Lopes, L. V. V071 Lord, H. P89 Lorenz, W. P58 Lottspeich, Friedrich P90 Ludewig, M. P102 Ludwig, Bernd P67 Lührmann, Reinhard P56, V024 Maaß, K. P41, P64 Macht, M. P21, P73 Maiolica, Alessio V082 Malik, A. K. P9 Mallick, Parag V074 Mandel, Friedrich P26, V054 Manea M. P122 Marcus, Katrin P63, P92, P94, V015 Marquis, A. P40 Martin Schuerenberg, Martin P36 Martin, C. V033, V065 Martin, Sue I. V095 Marvin-Guy, L. V071 Masterson, P. P12 Matter, U. V023 Matusch, Andreas P8, V011 May. M. P121 Mayer, Matthias P. P33 McIntyre, Douglas E. P26 McJimpsey, Erica L. V095 McKenna, T. P110, P111, P113 Meisen, Iris P65, V052 Meissner, Torsten V013 Melikyan, Anna P66 Metelmann-Strupat, W. V012 Metzger, Jürgen O. P28, P51, P52 Metzger, Sabine P55, P117 Meyer, Björn P67

Meyer, Helmut E.

P37, P63, P92, P94, V015, V114 Meyer, M. P68 Miekisch, W. P89 Mikkat, Stefan P69, P84, V072 Milosevic, Jadranka P17 Möder, M. P58, V033, V065 Möhring, Thomas V012 Moise, A. P70 Montgomery, H. P54, P121 Morgner, Nina V021 Moritz, Thomas P66 Mormann, M. P71, V044 Moumen, A. P12 Muck, Alexander V025 Müller, H. P101 Müller, Jutta V032 Müller, Thomas J. J. P77 Mundkowski, R. G. P89 Münster, Helmut V012 Muskat, Tassilo P72 Mutch, D. V071 Müthing, Johannes P65, V052 Neeson, Kieran P113 Neto, B. A. S. V124 Neubauer, York P107 Neubert, Reinhard P29, P82, P88 Neusüß, Christian P21, P118, V042 Nibbering, Nico M. M. W.-Paul-Vortrag Niebel, Joerg V055 Niemeyer, Dagmar P73 Nigge, Walter P43, P74 Noeldge-Schomburg, G. P27 Nolte, Andreas P63 Nolte, J. P5, P74, V034 Nowack, R. P68, V072 Oberemm, Axel P105 O'Connor, M. P12 Oppermann, J. P68, V072 Ostapczuk, P. P10 Otto, V. P70 Paizs, Béla P15 Palinsky, N. P102 Palomero-Gallagher, Nicola V011 Panchaud, A. V071 Paraschiv, Gabriela P75 Paschke, C. P41 Paschke, H. P58 Patricia, Fuchs P27 Pau, Hans Wilhelm P49 Pavam, C. H. V124 Pawliszyn, J. P89 Pelster, S. P76, V123 Pelzing, M. P21 Pelzing, Matthias P34, P77, V042 Peng, Song P43 Perkins, Patrick D. P26 Peschka, Manuela P79, P80

Peter-Katalinic, Jasna Pfeffer, B. Pick, U. Pickhardt, Carola Pieper, Stefan Pingoud, Vera Pitesky, Maurice E. Platzner, T. Poeaknapo, Chotima Pohlentz, G. Preiß, Alfred Preissner, Klaus T. Preywisch, Regina Prime, J. Pröbst, F. Prüfer, Ines Przybylski, Michael Raith, Klaus Rapp, U. Rappsilber, Juri Rauhut, Guntram Raymond, F. Reemtsma, Thorsten Reidegeld, Kai Reifenberger, Guido Reimer, T. Renne, U. Resch, Klaus Resch, M. Reuss, M. Richardson, Keith Richert, Joachim Riese, Mikko Riley, Timothy Ringel, Bruno Riot, Vincent J. Rist, Wolfgang Roepstorff, Peter Rohlfing, Andreas Römpp, Andreas Rontree, J. Rossier, Joel S. Rowold, Karsten Russel, Scott C. Rutzinger, S. Ruzsanyi, V. Sailer, Claudia Santos, L. S. Sauermann, S. Schaefer, Heike Schäfer, Jürgen Scheffler, K. Scheffner, M. Scherzer, N. Schiebel, Hans-Martin Schinke, I. Schirle, Markus

P14, P65, P71, P120, V041, V044, V052, V084 P23 P109 P10, P11, P6, P8, P9, V011 P81 P30 V095 P9 V125 P71, V044 V121 P17 P114 P12 P18 P49, P68, V072 P6, P7, P70, P75, P114, P122, V011 P82, P88 F09 V082 P57 V071 V031 P37, P116, V114 P8 P38 P115 P53 P121 V114 P113 P35 P83, P86 P113 P49, P54, P68, P84 V095 P33 HV2 V052 P85 F05 V041 P86 V095 P18 P3, P5, V061 V113 V124 P103 V015 V075 F04 P122 P89 V121 P84 V074

Schlosser, Andreas Schlosser, Dietmar Schmalz, Natalie Schmelzer, Christian E. H. Schmidt, Alexander Schmidt, Andrea Schmidt, Andreas Schmidt, Jürgen Schmidt, Oliver Schnelle-Kreis, J. Schöler, Heinz F. Schott, Mathias Schrader, S. Schrader, W. Schreiber, A. Schubert, J. Schubert, Jochen K. Schubert, K. Schulenborg, Thomas Schulte, Uwe Schulz, Daniela M. Schulz, Gary Schulz, O. Schulz-Tönnies, Karen Schümann, Michael Schürpf, T. Schüth, F. Schwarz, Josef Schwarz, Ludwig Schweiger, Stefan Schwudke, D. Šebela, Marek Seeger, Werner Seeling, U. Segal, I. Seidel, W. Sekiya, Sadanori Serrano-Fernandez, P. Shellie, R. Shevchenko, A. Shevchenko, Andrej Siddiqui, Khalid Siemann, M. Silva, Jeffrey C. Simon, Michael Sinz, Andrea Smyth, Neil Snel, M. Snel, Marten Sobek, Dan Sommer, H. Sonnenschein, H. Sparbier, Katrin Spengler, Bernhard Spiess, Joachim Springer, A. Springer, Michael Springer, Nicola

V103 V033 P87 P29, P82, P88 P90 P44, P56 P91, P95 V125 P92 V064 P34 P93 P58 P76, V123 V092 P89 P27 P21 P92, P94 V113 P91, P95 V055 P16 P107 P105 P70 P76, V123 V075 P99 P57 V062 P39, P87 P17 P9 P9 P18 V043 P38 V064 P109, V062 P39, P62, P87 P67 V114 P113 P119 P47, P50, P91, P95, V111 P50 P110, P111 V022 V054 F03 P2 P96 P1, P16, P41, P64, P85, V051, V083, V094 V112 P97 V101 P98

Srivastava, Abneesh Starke, I. Steele, Paul T. Steen, Hanno Steiner, Sandra Stephan, Michael Stößer, R. Strupat, Kerstin Sturre, Marcel J. G. Suckau, D. Suhai, Sindor Svatoš, Aleš Svoboda, Michal Sykora, C. Szadkowski, M. Szmigielski, R. Taban, Ioana Tampé, Robert Tanaka, Koichi Tenz, Kareen Teplyakov, N. These, Anja Thiele, Herbert Thiery, J. Thiery, Joachim Thiesen, H.-J. Thieu, Vinh An Tholey, Andreas Thomas, Henrik Thumfart, Jörg Oliver Tian, Xiaodan Tinazli, Ali Tobias, Herb J. Toennes, Stefan W. Turecek, Frantisek Uchaikin, S. Urfer, W. Urlaub, Henning Vakhrushev, S. Y. van de Goor, Tom van Dorsselaer, A. van Leeuwen, Fred van Pelt, Colleen Vana, P. Vanselow, Jens Varga, Z. Vautz, W. Vinkemeier, Uwe Vinken, R. Vintan, Rares von Czapiewski, Kristin Vukelic, Željka Wada, Yoshinao Wagner, Moritz Walbrodt, Dirk Wanczek, Karl-Peter Wangler, Tanja Waridel, P. Warnke, Jörg

V095 P101 V095 V101 V075 P102 P42 V012 P37 P36 P15 V025 P53, V091 V102 P12 P104 P85 P108 P54, P121, V043 P105 P9 V031 V121 V063 V073 P19, P68, P84, V072 P85, V083 P106 P39, P87 V113 P75 P108 V095 V093 HV1 P18 P4 P56, V024 V084 V054 P40 P94 V055 P25 V103 P10 P3, P5, V061 V013 V065 V035 P20, V091 P120, V052 V043 V093 P72 V122 V113 P109 P93

Warscheid, Bettina Weber, Reinhold Weinmann, W. Weiss, Anthony S. Weisser, M. Welthagen, W. Wenzel, Thomas Werner, Andreas Weyermann, C. Wiechert, M. R. Wiese, Sebastian Wiethölter, Nicola Wirth, H.J. Witt, Matthias Woisch, R. Wojciechowski, K. Wolf, Rainer Wolfram, Miekisch Wolkenstein, Klaus Yin, Hongfeng Youhnovski, Nikolay Young, Phillip Zabet-Moghaddam, Masoud Zamfir, Alina Zeller, Martin Zenk, Meinhart H. Zhang, Guangzu Ziegmann, Christian Zilles, Karl Zimmermann, D. Zimmermann, R. Zoriy, M. Zschörnig, Olaf

P37, P116, V114 P114 V092 P29 F10 V064 P96 P78 V094 P115 P116 P117 P23 P118 P19, P38, V023 P104 P35, P119 P27 P34 V054 P114 P113 P106 P14, P120, V041 V012 V125 P100 P36 P8, V011 P5, V061 V064 P10, P11, P6, P8, P9, V011 P95