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Mass Spectrometry Based Bioanalytics on Model Organisms

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Mass Spectrometry Based Bioanalytics on Model Organisms Mass Spectrometry based Bioanalytics on Model Organisms Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von Christian Treitz Kiel, 2017 Erster Gutachter: Prof. Dr. Andreas Tholey Zweiter Gutachter: Prof. Dr. Matthias Leippe Tag der mündlichen Prüfung: 22.02.2018 Zum Druck genehmigt: 22.02.2018 Danksagung An dieser Stelle möchte ich mich bei allen bedanken, die mit ihrer Unterstützung zu der Fertigstellung dieser Dissertation beigetragen haben. Mein herzlicher Dank gilt meinem Doktorvater, Professor Andreas Tholey, der mit seiner kompetenten Betreuung und unermüdlichen Motivation maßgeblich zur erfolgreichen Fertigstellung dieser Arbeit beigetragen hat. Das entgegengebrachte Vertrauen, die interessanten Projekte und den konstruktiven Austausch in zahlreichen wissenschaftlichen und persönlichen Gesprächen weiß ich sehr zu schätzen. Bei den jetzigen und auch ehemaligen Kollegen der Arbeitsgruppe möchte ich mich nicht nur für das positive Arbeitsklima sondern auch für die bereitwillige Anteilnahme an Freizeitaktivitäten und dem damit gelieferten Ausgleich bedanken. Für die Durchsicht, Korrektur und Diskussion meiner Arbeit bedanke ich mich bei Dr. Liam Cassidy, Dr. Diego Yepes, Dr. Tomas Koudelka, Jan Leipert und Patrick Kaleja. Insbesondere Liam hat einen Großteil der Arbeit korrekturgelesen und meine Vergehen an der englischen Sprache tapfer ertragen; thank you. Beim Thema Korrekturlesen darf ich auch Joanna Tucher nicht vergessen, die in der kurzen Zeit, zurück zu Besuch im hohen Norden, so lieb war die Zusammenfassung zu lesen. Des Weiteren möchte ich mich bei den zahlreichen Kooperationspartnern bedanken, ohne deren Hilfe die behandelten Forschungsprojekte nicht zustande gekommen wären. Dabei gilt mein Dank Professor Matthias Leippe und den Mitarbeitern des Zoologischen Instituts der Christian-Albrechts-Universität für das Konzept zur Erforschung erkrankter Fadenwürmer. Ohne die zahlreichen Ideen und richtungsweisenden Ratschläge zur Umsetzung des Projekts wäre es zweifellos nicht zu einem erfolgreichen Abschluss gekommen. Insbesondere bedanke ich mich auch bei der ehemaligen Mitarbeiterin Aylin Höckendorf für ihre Unterstützung bei der tagtäglichen (und nächtlichen), scharenweisen Anzucht und Ernte von Würmern. Den Freunden und Kollegen aus Toulouse, Brice Enjalbert, Fabien Létisse und Pierre Millard, die unter der Schirmherrschaft von Professor Jean Charles Portais forschen, möchte ich für die Gastfreundschaft im sonnigen Süden Frankreichs einen Tost aussprechen. Obwohl der kurze Forschungsaufenthalt der aeroben Kultivierung von Mikroben gewidmet war, erinnere ich mich mit besonderer Wehmut an die exzellenten Gärungsprodukte des Landes. Des Weiteren möchte ich mich bei Dr. Cleo Pietschke und Dr. Sebastian Fraune für meine Einbeziehung in ein weiteres sehr interessantes Forschungsprojekt, die erfolgreiche Kooperation und ihr persönliches Engagement bedanken. Tief verbunden und dankbar bin ich auch meinen Freunden und meiner Familie für ihre Unterstützung und ihr Verständnis dafür, dass sie in den letzten Jahren viel auf meine Anwesenheit verzichten mussten. Herzlichen Dank, Christian Treitz Lebenslauf Persönliche Daten Name: Christian Treitz Adresse: Olshausenstraße 7 24118 Kiel Mail: [email protected] Geburtsdatum: 25.05.1981 Geburtsort: Landau Staatsangehörigkeit: Deutsch Schulbildung 09 / 1991 – 06 / 2000: Realgymnasium Völklingen 06 / 2000: Allgemeine Hochschulreife Hochschulstudium 11 / 2001 – 11 / 2010: Diplomstudium Biologie Schwerpunkt Human- und Molekularbiologie Universität des Saarlandes 11 / 2003: Vordiplom 02 / 2004 – 07 / 2006: Laborassistent am Korea Institute of Science and Technology Europe 07 / 2009 – 10 / 2009: Diplomprüfungen Nebenfach: Genetik, Molekularbiologie Hauptfach: Biochemie 10 / 2009: Hilfswissenschaftler am Institut für Experimentelle Medizin (IEM), Arbeitsgruppe Systematische Proteomforschung, Christian-Albrechts-Universität zu Kiel (CAU) 12 / 2009 – 10 / 2010: Diplomarbeit zum Thema „Quantitative Analyse von Proteinen der Hefe Zellwand“ am IEM, AG Systematische Proteomforschung (CAU) seit 01 / 2011: Beginn des Promotionsstudiums am IEM, AG Systematische Proteomforschung (CAU) Publikationen Teile dieser Dissertation finden sich in folgenden Veröffentlichungen wieder: Treitz C, Cassidy L, Höckendorf A, Leippe M, Tholey A (2015). Quantitative proteome analysis of Caenorhabditis elegans upon exposure to nematicidal Bacillus thuringiensis. J Proteomics, 113: 336-350. Treitz C, Enjalbert B, Portais JC, Letisse F, Tholey A (2016). Differential quantitative proteome analysis of Escherichia coli grown on acetate vs. glucose. Proteomics, 16: 2742-2746. Pietschke C, Treitz C, Forêt S, Schultze A, Künzel S, Tholey A, Bosch T, Fraune S (2017). Host modification of a bacterial quorum sensing signal induces a phenotypic switch in bacterial symbionts. Proc Natl Acad Sci, 114: E8488-E8497, doi: 10.1073/pnas.1706879114. Tholey A, Treitz C, Kussmann M, Bendixen E, Schrimpf SP, Hengartner MO. (2013). Model organism proteomics - from holobionts to human nutrition. Proteomics, 13: 2537 - 2541. Mitwirkung an Veröffentlichungen zusätzlich zu den Teilen dieser Dissertation: Wilde SC, Treitz C, Keppler JK, Koudelka T, Kalpani K, Tholey A, Rawel HM, Schwarz K (2016). ß-Lactoglobulin as nanotransporter - part II: characterization of the covalent protein modification by allicin and diallyl disulfide. Food Chem, 197: 1022-1029. Leipert J, Treitz C, Leippe M, Tholey A (2017). Identification and quantification of N-acyl homoserine lactones involved in bacterial communication by small-scale synthesis of internal standards and matrix-assisted laser desorption/ionization mass spectrometry. J Am Soc Mass Spectrom, in press, doi: 10.1007/s13361-017-1777-x. Patent: Wilde SC, Keppler JK., Schwarz K. Palani K, Treitz C, Koudelka T, Tholey A (2017). Food additive comprising bio-active components of garlic google.com/patents/WO2017050314A1 Table of contents Table of contents Table of contents ............................................................................................................. 1 List of abbreviations ........................................................................................................ 5 Zusammenfassung …………………………………………………………………………….. 7 Summary ........................................................................................................................... 9 Chapter 1. General introduction 1.1 Bioanalytical research on model organisms ............................................................... 11 1.2 Proteomics sample preparation …………………………………………………………... 13 1.3 Protein and peptide separation ………………………………………………………….... 15 1.4 igh-performance liquid chromatography……………………………………………....... 18 1.5 Mass spectrometry……………………………………………………………………......... 22 1.6 Peptide identification………………………………………………………………….......... 24 1.7 'uantitative proteomics…………………………………………………………................ 26 Chapter 2. Quantitative proteome analysis of Caenorhabditis elegans upon exposure to nematicidal Bacillus thuringiensis 2.1 Abstract………………………………………………………………………………………. 30 2.2 Introduction…………………………………………………………………………….......... 32 2.3 Material and methods 2.3.1 Materials………………………………………………………………………............. 35 2.3.2 C. elegans cultivation and sample preparation…………………………………..... 35 2.3.2.1.1 ,ynchroni-ation by hypochlorite treatment……………………………... 35 2.3.2.1.2 ,accharose floating …………………………………………………........ 36 2.3.2.1.3 .orms sifting using nylon net filters…………………………………….. 36 2.3.2.1.4 /alidation of sample preparation procedures by microscopic survey.. 36 2.3.2.1.5 0ultivation of C. elegans and incubation 1ith Bacillus thuringiensis… 37 2.3.3 Protein e2traction, digestion and isobaric labeling………………………………... 38 2.3.4 T1o-dimensional liquid chromatography coupled to mass spectrometry………. 38 1 Table of contents 2.3.5 4ata processing………………………………………………………………………. 40 2.4 Results and discussion 2.4.1 0ultivation of C. elegans and sample preparation for proteomics………………. 41 2.4.1.1 Measurement of C. elegans dimensions by microscopy...……………... 43 2.4.1.2 /alidation of nylon net filter assisted sorting...…………………………... 44 2.4.1.3 ,ample preparation for C. elegans-pathogen interaction proteomics.... 45 2.4.2 'uantitative proteome analysis by isobaric labeling and 24-60 M, …………... 47 2.4.3 Proteins identified from pathogenic B. thuringiensis ……………………………... 53 2.4.4 Effects of pathogenic challenge on the C. elegans proteome…………………… 54 2.4.5 4ifferentially abundant protein families…………………………………………….. 55 2.5 0onclusion…………………………………………………………………………………… 62 Chapter 3. Differential quantitative proteome analysis of Escherichia coli grown on acetate vs. glucose 3.1 Abstract………………………………………………………………………………………. 64 3.2 Introduction…………………………………………………………………………………... 66 3.3 Material and methods 3.3.1 0ultivation of E. coli………………………………….…………..……………........... 68 3.3.2 Proteomics sample preparation………………………………..……………………. 68 3.3.3 Peptide fractionation by high p reversed-phase P60……………………….... 69 3.3.4 60-M, analysis of pooled fractions..................................................................... 69 3.3.5 4ata analysis....................................................................................................... 71 3.3.6 4ata visuali-ation and integration 1ith e2ternal data sources............................. 73 3.4 Results and discussion 3.4.1 4ifferential cultivation………………………………………………………………… 74 3.4.2 24-60-M,
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