DISSERTATION / DOCTORAL THESIS Titel der Dissertation / Title of the Doctoral Thesis “In-depth mass spectrometry-based proteome profiling of inflammation-related processes” verfasst von / submitted by Andrea Bileck, MSc angestrebter akademischer Grad / in partial fulfilment of the requirements for the degree of Doktorin der Naturwissenschaften (Dr.rer.nat.) Doctor of Sciences (Dr.rer.nat.) Wien, 2016 / Vienna 2016 Studienkennzahl lt. Studienblatt / degree programme code as it appears on the student A 796 605 419 record sheet: Dissertationsgebiet lt. Studienblatt / Chemie / Chemistry field of study as it appears on the student record sheet: Betreut von / Supervisor: Univ. Prof. Dr. Christopher Gerner II This doctoral thesis is based on the following publications and manuscripts: Comprehensive assessment of proteins regulated by dexamethasone reveals novel effects in primary human peripheral blood mononuclear cells Andrea Bileck, Dominique Kreutz, Besnik Muqaku, Astrid Slany, Christopher Gerner Journal of Proteome Research, 2014, 13, (12), 5989-6000 Impact of a synthetic cannabinoid (CP-47,497-C8) on protein expression in human cells: evidence for induction of inflammation and DNA damage Andrea Bileck, Franziska Ferk, Halh Al-Serori, Verena J. Koller, Besnik Muqaku, Alexander Haslberger, Volker Auwärter, Christopher Gerner, Siegfried Knasmüller Archives of Toxicology, 2015, doi:10.1007/s00204-015-1569-7 Shotgun proteomics of primary human cells enables the analysis of signaling pathways and nuclear translocations related to inflammation Andrea Bileck, Rupert L. Mayer, Dominique Kreutz, Tamara Weiss, Astrid Slany, Christopher Gerner Journal of Proteome Research, 2015, manuscript under revision Contribution of human fibroblasts and endothelial cells to the hallmarks of inflammation as determined by proteome profiling Astrid Slany, Andrea Bileck, Dominique Kreutz, Rupert L. Mayer, Besnik Muqaku, Christopher Gerner Molecular & Cellular Proteomics, 2016, manuscript in press III IV Acknowledgements First and foremost, I want to express my sincere gratitude to my supervisor Univ.-Prof. Dr. Christopher Gerner. I especially want to thank him for giving me the great opportunity to perform my doctorate in his group as well as for his guidance, valuable support and for a lot of fruitful and inspiring discussions during this time. His tremendous enthusiasm and immense knowledge really motivated me to give my best and to learn something new every single day. I would like to give my sincere thanks to Dr. Astrid Slany helping and supporting me in every stage of my doctorate. I especially want to thank her for always willing to listen to me, her valuable advice and loyalty. Furthermore, I would like to thank my colleagues, which have become really good friends, Rupert Mayer, MSc, Mag. Dominique Kreutz and Mag. Günter Walder for their great support, advice and all the fun we had in the last years. I want to express my sincere gratitude also to Dr. Samuel Meier. I especially want to thank him for his tremendous support in the final phase of my doctorate. In addition, I want to thank all the other members of our working group, Ammar Tahir, MSc, Besnik Muqaku, MSc, Mag. Denise Wolrab, Peter Frühauf and Dr. Johanna Mader for their support, great discussions and the good working atmosphere. I would also like to express my gratitude to my former colleagues Dr. Michael Grössl and Editha Bayer for introducing me in the field of mass spectrometry and sample preparation, as well as to our diploma students, especially Raphael Ambros for their help and assistance. Last but not least, I want to thank my whole family and friends, especially my mother Herta Bileck, for their invaluable support, motivation, understanding and patience on every day of my life. V VI Table of Contents 1. Abbreviations ............................................................................................................... p. IX 2. Introduction ................................................................................................................. p. 1 2.1. Immune System and Inflammation ........................................................................... p. 1 2.2. Chronic Inflammation and Cancer ........................................................................... p. 4 2.3. Therapeutic Modulation of Inflammation ................................................................ p. 7 2.4. Mass Spectrometry-based Proteomics ...................................................................... p. 9 2.4.1. General Overview of Mass Spectrometry-based Proteomics .............................. p. 9 2.4.2. Gel-based Proteomics .......................................................................................... p. 10 2.4.3. Gel-free Proteomics ............................................................................................. p. 11 2.4.4. Ionization Sources in Mass Spectrometry-based Proteomics .............................. p. 11 2.4.5. Mass Analyzers Commonly Used in the Field of Proteomics ............................. p. 12 2.4.6. Ion Activation Methods in Mass Spectrometry-based Proteomics ..................... p. 14 2.5. General Overview of Applied Methods in this Doctoral Thesis .............................. p. 15 2.5.1. Isolation and Treatment of PBMCs ..................................................................... p. 15 2.5.2. Subcellular Fractionation ..................................................................................... p. 16 2.5.3. Sample Preparation for Mass Spectrometric Analysis ........................................ p. 16 2.5.4. LC-MS/MS Analysis ........................................................................................... p. 17 2.6. Challenge of Secretome Analysis ............................................................................. p. 18 2.7. High Data Density Obtained by Mass Spectrometry-based Proteomics .................. p. 20 2.8. Challenges in the Analysis of PTMs using Mass Spectrometry ............................... p. 22 2.9. Peptide Identification and False Discovery Rate Estimation ................................... p. 23 2.10. Research Justification ............................................................................................... p. 24 2.11. References ................................................................................................................ p. 29 3. Results and Discussion ................................................................................................ p. 33 3.1. Comprehensive Assessment of Proteins Regulated by Dexamethasone Reveals Novel Effects in Primary Human Peripheral Blood Mononuclear Cells .................. p. 33 3.2. Impact of a Synthetic Cannabinoid (CP-47,497-C8) on Protein Expression in Human Cells: Evidence for Induction of Inflammation and DNA Damage ............. p. 47 3.3. Shotgun Proteomics of Primary Human Cells Enables the Analysis of Signaling Pathways and Nuclear Translocations Related to Inflammation .............................. p. 63 3.4. Contribution of Human Fibroblasts and Endothelial Cells to the Hallmarks of Inflammation as Determined by Proteome Profiling ................................................ p. 97 4. Conclusions................................................................................................................... p. 143 5. Abstract ........................................................................................................................ p. 145 6. Zusammenfassung (German Abstract) ..................................................................... p. 146 7. Scientific Contributions ............................................................................................. p. 147 7.1. List of Publications ................................................................................................... p. 147 7.2. List of Oral Contributions ........................................................................................ p. 148 7.3. List of Poster Contributions ...................................................................................... p. 148 VII VIII 1. Abbreviations 2D-DIGE, 2-dimensional difference gel electrophoresis; 2D-PAGE, 2-dimensional polyacrylamide gel electrophoresis; ACN, acetonitrile; AP-1, transcription factor AP-1; APCs, antigen-presenting cells; BM, basement membrane; CID, collisional-induced fragmentation; COX, cyclooxygenase; CTLs, cytotoxic T cells; DAMP, damage-associated molecular pattern; DCs, dendritic cells; DTT, dithiothreitol; ECs, endothelial cells; EI, electron ionization; ESI, electrospray ionization; ETD, electron transfer dissociation; FA, formic acid; FASP, filter-aided sample preparation; FCS, fetal calf serum; FDR, false discovery rate; GC, glucocorticoid; GRE, glucocorticoid response element; HCD, higher energy collisional dissociation; HIF1α, hypoxia-inducible factor 1α; IAA, iodoacetamide; ICAM-1, intracellular adhesion molecule-1; IL, interleukin; IPGs, immobilized pH gradients; LC, liquid chromatography; LOX, lipoxygenase; LPS, lipopolysaccharide; MALDI-TOF, matrix-assisted laser/desorption ionization-time of flight; MMPs, matrix metalloproteinases; MRM, multiple reaction monitoring; MS, mass spectrometry; MWCO, molecular weight cut-off; NF-κB, nuclear factor- κB; NK cells, natural killer cells; NSAIDs, non-steroidal antiphlogistic drugs; PAMP, pathogen- associated molecular pattern; PBMCs, peripheral blood mononuclear cells; PG, prostaglandin; PHA, phytoheamagglutinin; PMF, peptide