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Proteomics of Prostate Proximal Fluids to Guide Biomarker Discovery

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Proteomics of Prostate Proximal Fluids to Guide Biomarker Discovery Proteomics of Prostate Proximal Fluids to Guide Biomarker Discovery by Katharina Fritsch A thesis submitted in conformity with the requirements for the degree of Master of Science Medical Biophysics University of Toronto © Copyright by Katharina Fritsch 2019 Proteomics Panorama of Prostate Proximal Fluids Katharina Fritsch Master of Science Medical Biophysics University of Toronto 2019 Abstract Prostate Cancer is the most common non-skin cancer in men. Current diagnostic factors are inaccurate in predicting outcome, resulting in over- and undertreatment of many men. Improved prognostic factors that enable non-invasive diagnosis and follow-up are strongly needed. I hypothesize that comparative proteomic profiling of a direct EPS cohort will identify prognostic biomarkers. I developed and applied a shotgun proteomics assay to a cohort of 148 clinically stratified direct EPS samples. These analyses identified 1271 peptides that are significant between risk categories. Results were independently verified in a direct EPS cohort from Virginia with 115 samples (59 overlap). 228 of 1271 peptides showed the same trend over risk groups in both datasets. Putative biomarkers will be validated using targeted proteomics assays in an independent cohort of post-DRE urines. In the future these assays could assist to accurately stratifying patients into prostate cancer risk groups. ii Acknowledgments This project and my interest in research would not be possible without the influences of my past and present supervisors, colleagues and professors. Firstly, I would like to thank my supervisor Thomas Kislinger for the opportunity to work in his proteomics laboratory at Princess Margaret Cancer Centre, Toronto, his guidance and support in conducting my Master project. I am thanking my committee members Stanley Liu and Paul Boutros for their important input and guidance throughout this project and their expertise in research, especially in prostate cancer and data analysis. Additionally, I would like to thank John O. Semmes and Julius O. Nyalwidhe from the Eastern Virginia Medical School in Norfolk, Virginia, US for sending us the clinically stratified patient samples. Secondly, I would like to thank my coworkers in the Kislinger lab and my fellow students from the Medical Biophysics department at the University of Toronto for their help and support. Thirdly, I am grateful towards my family and friends in Munich, who supported me from far away and stayed connected with me over the last two years living on a different continent. I am so thankful for all the amazing people I met in Toronto and get to do life with – I would not want it any other way. Lastly, I am thanking God for all that He has given and entrusted to me (including my brain). I thank Him for always helping me to grow and flourish through the highs and the lows. For showing me that deep truth that exceeds everything, for knowing me better than any human being (including myself) and loving me the way I am and constantly healing my soul. For being the light, truth, and peace when my thoughts wage war. And for filling me with joy in every season no matter what it may bring. ‘But blessed is the one who trusts in the Lord, whose confidence is in Him. They will be like a tree planted by the water that sends out its roots by the stream. It does not fear when heat comes; its leaves are always green. It has no worries in a year of drought and never fails to bear fruit.’ Jeremiah 17:7-8 NIV iii Table of Contents 1. Introduction ........................................................................................................................... 1 1.1 The prostate gland .......................................................................................................... 1 1.2 Prostate cancer ............................................................................................................... 3 1.2.1 Prostate cancer statistics and risk factors ................................................................. 3 1.2.2 Histopathology of prostate cancer ............................................................................ 4 1.2.3 Current diagnosis and risk stratification of prostate cancer ...................................... 7 1.3 Prostate Cancer Biomarkers ......................................................................................... 14 1.3.1 Introduction to cancer biomarkers ........................................................................... 14 1.3.2 Biomarkers in tissue proximal fluids and biological fluids ....................................... 15 1.4 Shotgun proteomics for biomarker discovery ................................................................ 17 1.5 Hypothesis and Aims ..................................................................................................... 21 2. Materials and Methods ........................................................................................................ 22 2.1 Materials ........................................................................................................................ 22 2.2 Methods ........................................................................................................................ 23 2.2.1 Sample Cohort ........................................................................................................ 23 2.2.2 Bicinchoninic Acid Assay ........................................................................................ 24 2.2.3 Trypsin Digestion using MStern Blotting ................................................................. 25 2.2.4 Solid Phase Extraction ............................................................................................ 26 2.2.5 Liquid Chromatography and Mass Spectrometry .................................................... 26 2.2.6 Analysis of Mass Spectrometry Data ...................................................................... 28 2.2.7 Gene Ontology Pathway Analysis ........................................................................... 28 2.2.8 Differential Expression Analysis .............................................................................. 28 3. Results ................................................................................................................................ 30 3.1 Method Optimization ..................................................................................................... 30 iv 3.1.1 MStern Digestion .................................................................................................... 30 3.1.2 High throughput SPE in 96 well format ................................................................... 32 3.1.3 Internal standards SUC2 and iRT ........................................................................... 33 3.2 Risk group placement .................................................................................................... 35 3.3 Data analysis ................................................................................................................. 38 3.3.1 Data quality check .................................................................................................. 38 3.3.2 Comparison to previously published datasets......................................................... 45 3.3.3 Data overview (protein numbers, GO analysis) ...................................................... 47 3.3.4 Differentially abundant peptides .............................................................................. 51 4. Comparison to Independent Direct EPS Data ..................................................................... 62 4.1 Data Quality for Virginia Cohort ..................................................................................... 62 4.2 Qualitative Comparison of Toronto and Virginia Data ................................................... 66 4.3 Filtering for Peptides ..................................................................................................... 68 5. Discussion .......................................................................................................................... 72 6. Outlook ............................................................................................................................... 74 7. Abbreviation and Symbols .................................................................................................. 76 8. References ......................................................................................................................... 80 9. Supplemental Figures ......................................................................................................... 92 v 1. Introduction 1.1 The prostate gland The prostate is a small exocrine gland in the male reproductive system. It contributes secreted proteins to the seminal fluid to keep sperm alive and sustain their mobility while protecting the genetic code they carry1. The gland is located at the base of the bladder in front of the rectum surrounding the urethra (Figure 1) and is comprised of 70% glandular and 30% fibromuscular or stromal tissue2. Figure 1: Schematic of the male urogenital tract. McNeil established the commonly accepted concept of various zones of the prostate gland with different probabilities to develop carcinomas3 (Figure 2). The peripheral zone constitutes up to 70% of the gland and comprises the prostatic glandular tissue at the apex. It shows the highest probability to give rise to carcinoma (70-80%), post-inflammatory atrophy,
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