PALACKÝ UNIVERSITY FACULTY OF MEDICINE AND DENTISTRY Program DSP: Oncology CANCER PROTEOMICS IN CLINICAL AND EXPERIMENTAL ONCOLOGY Tomáš Oždian, M.Sc. Supervising department: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University and University Hospital in Olomouc Supervisor: Petr Džubák, MD, PhD Olomouc 2016 I hereby declare that this thesis has been written solely by myself and that all the sources used in this thesis are cited and included in the references part. The research was carried out in the frame of the Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University Olomouc. In Olomouc ……………………… September 2016 Tomáš Oždian, M.Sc. Acknowledgments I would like to thank to my supervisor, Petr Džubák, M.D., Ph.D. for his valuable advices, willingness and help. My thanks belong also to Marián Hajdúch, M.D., Ph.D. for facilitating the laboratory work at the Institute of Molecular and Translational Medicine, with the great advantage of state of the art equipment and guidance throughout the research. I would like to thank to all my colleagues from IMTM Olomouc, especially Lakshman Varanasi, who helped me with writing of articles and dissertation. My appreciation goes to my colleagues from the Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacký University, Olomouc for great collaboration. Last, but not at least I would like to thank my family and friends for their patience and motivation during all time of my study. Research on these projects was supported by grants: CK TAČR TE02000058, LF_2016_019 and National Sustainability Programme (LO1304). Olomouc …………………… September 2016 Tomáš Oždian, M.Sc. Bibliografická identifikace Jméno a příjmení autora Tomáš Oždian Název práce Nádorová proteomika v klinické a experimenální onkologii Typ práce Dizertační Pracoviště Ústav molekulární a translační medicíny, Lékařská fakulta, Univerzita Palackého v Olomouci Vedouci práce MUDr. Petr Džubák, Ph.D Rok obhajoby práce 2016 Klíčová slova Proteomika, hmotnostní spektrometrie, cisplatina, karboplatina, oxaliplatina, asporin, Jazyk Anglický Abstrakt Tato práce je zaměřena na proteomiku a její metody využívané při výzkumu léčiv a biomarkerů. V teoretické části jsou shrnuty hlavní body pracovních a analytických postupů moderní proteomiky. Experimentální část je rozdělena mezi dva projekty, které společně využívají většinu metod popsaných v teoretické části. První část diskutuje projekt proteomického profilování nádorových buněčných linií pro zjištění jejich odpovědi na léčivo. K tomu bylo využito více hmotnostně spektrometrických přístupů Proteomická analýza v tomto projektu odhalila, že hlavní buněčnou odpovědí na ošetření oxaliplatinou je nukleolární stres. Druhým projektem je přehled kvantitativních MS metod použitých k analýze hladiny potencionálního biomarkeru, asporinu. Specifická citlivost většiny metod na asporin byla velmi nízká. Důvody pro tuto nízkou citlivost jsou v této práci také diskutovány, nejpravděpodobnější je rezistence asporinu k trypsinové digesci. 4 Bibliographical identification: Author’s name and surname Tomáš Oždian Title Cancer proteomics in clinical and experimental oncology Type of thesis Dissertation Department Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University Olomouc Supervisor Petr Džubák, MD, PhD The year of presentation 2016 Keywords Proteomics, mass spectrometry, cisplatin, carboplatin, oxaliplatin, asporin Language English Abstract The thesis is focused into proteomic methods and their application in drug and biomarker research. Theoretical part reviews all major aspects of modern proteomic workflow and analysis. Experimental part is split into two parts covering together the large part of methods discussed in theoretical introduction. The first part discusses project of drug response determination by cancer cell line proteomic profiling. A suitability of different MS approaches for this kind of analysis is discussed here as well as successful determination of nucleolar stress as main response to oxaliplatin treatment. The second project of this thesis is an overview of MS quantification approaches in analysis of asporin protein. This protein showed itself to be particularly hard to analyse with reasonable sensitivity. The reasons of such low sensitivity specific to asporin are discussed here as well. The most probable reason is a resistance of asporin to trypsin digestion. 5 TABLE OF CONTENTS 1 INTRODUCTION ..................................................................................................8 1.1 Protein separation methods .............................................................................9 1.1.1 Electrophoretic methods ................................................................................. 9 1.1.2 Chromatographic methods ............................................................................ 13 1.1.3 Affinity purification ........................................................................................ 18 1.1.4 Precipitation ................................................................................................... 21 1.2 Protein identification methods ....................................................................... 23 1.2.1 Antibody based identification methods ......................................................... 24 1.2.2 Mass spectrometry based methods ............................................................... 29 1.2.3 Protein sequencing methods ......................................................................... 37 1.3 Top-down and bottom-up proteomics ............................................................. 38 1.3.1 Sample preparation and digestion in bottom-up proteomics ....................... 39 1.3.2 LC-MS analysis ................................................................................................ 42 1.3.3 MS based quantification ................................................................................ 44 1.3.4 Data processing and database search ............................................................ 49 1.3.5 Bioinformatic evaluation of results ................................................................ 50 1.4 Other MS-based proteomic methods .............................................................. 52 1.4.1 Targeted MS methods .................................................................................... 52 1.4.2 Non-covalent mass spectrometry .................................................................. 52 1.4.3 Surface enhanced laser desorption/ionisation .............................................. 53 2 EXPERIMENTAL PART ......................................................................................... 57 2.1 Aims .............................................................................................................. 57 2.2 Proteomic profiling of anticancer drugs ........................................................... 58 2.2.1 Material and methods.................................................................................... 59 2.2.2 Results ............................................................................................................ 65 2.2.3 Discussion ....................................................................................................... 81 2.3 Determination of asporin in breast cancer derived cell cultures. ...................... 87 6 2.3.1 Material and methods.................................................................................... 88 2.3.2 Results ............................................................................................................ 93 2.3.3 Discussion ..................................................................................................... 101 3 SUMMARY .................................................................................................... 104 4 SOUHRN ....................................................................................................... 107 5 REFERENCES .................................................................................................. 110 6 ABBREVIATIONS ............................................................................................. 128 7 BIBLIOGRAPHY ............................................................................................... 131 7.1 Original articles and reviews ......................................................................... 131 7.2 Oral and poster presentations ...................................................................... 131 8 APPENDIX - FULL TEXT PUBLICATIONS RELATED TO THE THESIS ..................................... 135 8.1 Appendix A .................................................................................................. 135 8.2 Appendix B ................................................................................................... 153 8.3 Appendix C ................................................................................................... 158 7 1 INTRODUCTION Proteomics is currently well-established method of bioscience. Basically, proteomics allows studying proteins in detail or in large scale identification experiments. Both approaches have their substantial merits. Knowledge of detailed protein properties is essential in deciphering protein function, activity or in description of protein inhibition by various drugs. Second approach, large scale