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Quantitative Proteome Analysis of Human Neuroblastoma Cells Treated with Clostridium Botulinum C3 Exoenzyme

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Quantitative Proteome Analysis of Human Neuroblastoma Cells Treated with Clostridium Botulinum C3 Exoenzyme Quantitative proteome analysis of human neuroblastoma cells treated with Clostridium botulinum C3 exoenzyme Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades einer Doktorin der Naturwissenschaften Dr. rer. nat. genehmigte Dissertation von Diplom Chemikerin Marika Victoria Mützelburg geboren am 16.05.1982 in Eisenhüttenstadt -2008- Referent: Prof. Dr. Thomas Scheper Koreferent: PD Dr. Andreas Pich Tag der Promotion: 01.09.2008 Die vorliegende Arbeit wurde in der Zeit von Juli 2005 bis Mai 2008 im Institut für Technische Chemie und im Institut für Toxikologie der Medizinischen Hochschule Hannover unter der Betreuung von Prof. Dr. T. Scheper, PD Dr. A. Pich und Prof. Dr. I. Just angefertigt. Erklärung Eidesstattliche Erklärung Ich erkläre an Eides statt, dass ich die hier vorliegende Arbeit selbständig und ohne fremde Hilfe verfasst habe. Ich habe dazu keine weiteren, als die hier aufgeführten Hilfsmittel benutzt und die aus anderen Quellen entnommenen Stellen als solche gekennzeichnet. Ich habe die Dissertation nicht als Diplomarbeit oder ähnliche Arbeit verwendet und abgesehen von den angegebenen Teilpublikationen nicht veröffentlicht. Marika Victoria Mützelburg Hannover, den 01.09.2008 I Danksagung Danksagung Allen, die mir mit Rat und Tat bei der Durchführung, sowie Anfertigung meiner Dissertation behilflich waren, möchte ich an dieser Stelle meinen herzlichsten Dank aussprechen. In erster Linie gilt mein Dank Herrn PD Dr. Andreas Pich und Herrn Prof. Dr. Ingo Just, die nicht nur die Grundidee zu dieser Arbeit lieferten, sondern mich auch förderten und mir jederzeit und im hohen Maße hilfreich zur Seite standen. Besonderer Dank geht an Karin Agternkamp und Zoltán Czentnár für die kraftvolle Unterstützung während der Laborarbeiten, sowie dem Beistand beim Kampf mit und gegen die Geräte. Für den ständigen Zufluss an Zellen und für die Durchführung der Vergiftungen - ein großes DANKE an Sandra Hagemann und PD Dr. Fred Hofmann. Diesem, sowie Zoltán Czentnár möchte ich an dieser Stelle noch einmal explizit für die Hilfestellung und die besonnenen Ratschläge Computer- und Systemtechnik betreffend danken. Da die Aufzählung aller Mitglieder des Institutes für Toxikologie den Rahmen sprengen würde, seien Corinna Mühlenstädt, Janett Schöntaube und Alexandra Olling hier besonders hervorgehoben. Sie halfen mir bei der Durchführung von Brainstorming Attacken und zeigten mir, dass es auch noch ein Leben neben der Dissertation und Arbeit gibt. Die höchstmögliche Anerkennung gilt den wichtigsten Menschen in meinem Leben: Daniel Jocksch, meinen Eltern Irina und Joachim Mützelburg, meinen Großeltern Jutta und Gerhard Schmidt, meinem Bruder Tom Mützelburg sowie meinen künftigen Schwiegereltern Gabriele und Siegfried Jocksch. II Widmung Diese Arbeit widme ich Daniel Jocksch, meinen Eltern und meinen Großeltern, welche mich während meines gesamten Studiums und der dreijährigen Promotionszeit in allen Bereichen des Lebens ohne Einschränkungen unterstützt haben. III Publikationen Publikationen Wissenschaftliche Publikationen Alnajjar, A., Idris, A.M., Multzenberg, M., McCord, B. (2007) Development of a capillary electrophoresis method for the screening of human urine for multiple drugs of abuse. J Chromatogr B Analyt Technol Biomed Life Sci. 856, 62-67. (Schreibfehler) Muetzelburg, M.V., Hoffmann, R. (2008) Separation of multiphosphorylated peptide isomers by capillary zone electrophoresis. Submitted to Electrophoresis (review process) Teile aus dieser Arbeit werden aus Prioritätsgründen veröffentlicht. Muetzelburg, M.V., Hofmann, F., Hahner, S., Just, I., Pich, A. (2008) Quantitative proteome analysis of human neuroblastoma cells treated with Clostridium botulinum C3 exoenzyme – application of isotope-coded protein labels and LC-MALDI techniques. Manuscript submitted for publication in Mol Cell Proteomics Muetzelburg, M.V., Hofmann, F., Just, I., Pich, A. (2008) Isobaric tags for relative and absolute quantitation coupled with 2D-LC-MALDI-MS/MS techniques enable the identification of biomarkers of C3 exoenzyme treated human neuroblastoma cell lines. Manuscript submitted for publication in J Chromtogr B. Muetzelburg, M.V., Czentnár, Z., Luecke, N., Just, I., Pich, A. (2008) Protein profiling of complex protein mixtures – A comparison between LC-MALDI-MS/MS and CE- ESI-MS/MS techniques. Manuscript in preparation Luecke, N., Templin, C., Kotlarz, D., Muetzelburg, M.V., Just, I., Pich, A. (2008) Secretom analysis of the hematopoethic progenitor cell line DKmix using LC-MALDI techniques. Manuscript in preparation IV-1 Publikationen Poster Alnajjar, A., Muetzelburg, M., Butcher, J., McCord, B. (2004) Determination of multiple drugs of abuse in human urine using capillary electrophoresis with fluorescence detection. Studiertenkolleg, Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio, USA Muetzelburg, M.V., Hoffmann, P., Singer, D., Hoffmann, R. (2005) Separation of phosphopeptide isomers using capillary-zone-electrophoresis. Biotechnology Symposium BBZ in Leipzig, Anakon und Beckman Coulter User Meeting (zweimal Posterpreis) Muetzelburg, M.V., Muehlenstaedt, C., Just, I., Pich, A. (2008) Isotope-coded protein labels and LC-MALDI techniques used for quantitative proteome analysis of intoxicated human neuroblastoma cells. Tagung der Deutschen Gesellschaft für Massenspektrometrie (DGMS) Muetzelburg, M.V., Muehlenstaedt, C., Just, I., Pich, A. (2008) Toxicoproteomics: C3 exoenzyme affects protein profile of a human neuroblastoma cell line. Tagung der Deutschen Gesellschaft für experimentelle und klinische Pharmakologie und Toxikologie (DGPT) Vorträge 11-2007 Vortrag im Rahmen des Massenspektrometrie Forums Hannover „Heavy Isotopes for Quantitative Proteomics“ 12-2006 Vortrag im Rahmen der Veranstaltung Basic Biological Questioner an der MHH „Quantitative Proteomics using ICPL™“ 09-2005 eingeladener Vortrag auf dem Beckman Coulter User Meeting „Trennung isomerer Phosphopeptide mittels Kapillarzonenelektro- phorese“ 07-2004 Vortrag im Rahmen des Studierendenkollegs am Department of Chemistry and Biochemistry der Ohio University, Athens, Ohio, USA „Derivatization of amine-containing drugs using Cy5™-NHS monoreactive Dye for capillary electrophoresis and microfluidic devices” IV-2 Zusammenfassung Zusammenfassung C3 Exoenzyme sind ADP-Ribosyltransferasen, die spezifisch die Rho-GTPasen RhoA, B und C modifizieren und damit inaktivieren. Rho-GTPasen sind molekulare „Schalter“, welche eine Vielzahl von Signalwegen in eukaryotischen Zellen kontrollieren. Die spezifische Inaktivierung der Rho-GTPasen führt zu einer Reorganisation des Aktinzytoskeletts und aller aktinabhängigen Zellfunktionen. In den letzten Jahren konnte eine Beteiligung der Rho-GTPasen an der neuronalen Entwicklung gezeigt werden. Die Zugabe von C3 aus Clostridium botulinum (C3bot) zu Hippocampuszellen resultiert in einem gesteigerten Auswachsen und einer verstärkten Verzweigung der Axone. Zur Aufklärung dieser C3bot-induzierten Wirkungen wurde mittels eines quantitativen Proteomic-Ansatzes das Proteinprofil von C3bot-behandelten mit unbehandelten humanen Neuroblastomzellen (SH-SY5Y) verglichen. Dazu wurden die zellulären Proteine mit „Isotope Coded Protein Label“ (ICPL™) bzw. die nach tryptischer Spaltung erhltenen Peptide mit „isobaric tags for relative and absolute quantification“ (iTRAQ™) markiert, mittels Gelelektrophorese- und Flüssigchromatographie-basierter Techniken getrennt und massenspektrometrisch analysiert. In ersten Experimenten wurde ein Proteinprofil der SH-SY5Y Zellen mittels GeLC- MS/MS dargestellt und die Methodik optimiert. Mit der Reduzierung der Probenkomplexität auf Proteinebene konnten die Identifizierungsraten gesteigert und bis zu 3399 verschiedene Proteine identifiziert werden. Bislang existieren kaum Angaben über die Reproduzierbarkeit quantitativer Techniken für biologische Systeme. Daher wurde das ICPL™-Markierungsexperiment doppelt durchgeführt und eine durchschnittliche Variation von unter 30% bestimmt. Auf diese Weise konnten 1466 Proteine identifiziert werden, von denen 777 mit ICPL™ markiert waren. Zur ICPL™- Technik wurde alternativ die iTRAQ™-Methodik eingesetzt. In diesem Ansatz wurde das komplette Proteom zunächst tryptisch gespalten und dann modifiziert. Es wurden 355 Proteine identifiziert, von denen 235 mit iTRAQ™ markiert und quantifiziert werden konnten. Die extrem hohe Probenkomplexität des Peptidgemisches war verantwortlich für die im Vergleich zum ICPL™-Ansatz zu geringere Identifizierungsrate. Insgesamt wurden 1774 verschiedene Proteine in beiden Experimenten identifiziert, von denen 417 Proteine, durch C3bot-Behandlung in ihrer Konzentration signifikant verändert wurden. Diese Proteine sind im Wesentlichen an zellulären Wachstums- prozessen, DNA-/RNA-Prozessierung sowie Zellzyklus und Organisation des Aktinzytoskeletts beteiligt. Die erhaltenen Resultate lieferten neue Ansatzpunkte für die weitere Erforschung der Wirkung des C3bot Exoenzyms auf neuronale Zellen. Schlagworte: quantitative Proteomanalyse, Massenspektrometrie, C3bot Exoenzym V Abstract Abstract The C3 exoenzymes are ADP-ribosyltransferases, which inactivate the GTPases RhoA, B, and C. Rho-GTPases are molecular switches that control a variety of signal transduction pathways in eukaryotic cells. The specific inactivation of Rho-GTPases results in reorganization of the actin cytoskeleton. Evidence that neuronal growth depends on inactivation of Rho-GTPases arises in the last years. Recently, it
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