Enrichment and Identification of Methylation at the Proteome Level Alexandra Star A thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Degree of Master of Science in Biochemistry Department of Biochemistry, Microbiology, and Immunology Faculty of Medicine University of Ottawa ©Alexandra Star, Ottawa, Canada, 2016 ABSTRACT Methylation is a post-translational modification which occurs on lysine and arginine residues. Methylation is difficult to detect due to its low abundance and lack of charge. Our laboratory previously developed a novel enrichment approach, ProMENADe, for lysine and arginine methylation in the human embryonic kidney (HEK) 293T cell line which is coupled with mass spectrometry. Simplifying a lysate with subcellular fractionation prior to enrichment increased the identification of methylation sites by 39.5% while using multiple proteases for digestion increased identification by 27%. Combining these methods yielded a 47.2% increase. Analysis at the 1% methylation level FDR filtered for C-terminal methylation identified 169 sites and further analysis revealed 74 of these sites overlap with the PhosphoSite database. This ProMENADe enrichment strategy yielded 95 novel methylation sites to the field and can be a key tool in the field of methylation allowing for the enrichment and identification of methylated proteins. ii ACKNOWLEDGEMENTS I would like to thank Dr. Daniel Figeys for giving me the opportunity to study under his guidance and the guidance of Dr. Jean-François Couture. Over the past two years I have learned about proteomics, mass spectrometry, and the difficulties in studying post- translational modifications like methylation. Dr. Figeys gave me the opportunity to be independent, and for that I am grateful. I would also like to acknowledge my thesis advisory committee members Dr. Alexandre Blais and Dr. Jocelyn Côté for their guidance and re-direction in my project. I would also like to thank Deeptee Seebun for training me in the lab. I would like to acknowledge Dr. Janice Mayne for motivating and supporting me throughout my masters and for meeting with me weekly to discuss experiment ideas and working tirelessly to edit all of my TAC reports, presentations and my thesis itself. I am grateful to Dr. Zhibin Ning whose method has made my project possible and for all of his discussion and scientific advice. Although we work differently, he has helped me change the way I approach problems and has challenged me throughout my learning process. Lastly I would like to thank Dr. Sylvain Lanouette for the many discussions in regards to methylation and SMYD2, and for the guidance in writing my thesis I would also like to thank my lab mates Deeptee Seebun, Shelley Deeke, Dr. Amanda Starr, Myriam Cramet, Kiara Chu, Dr. Bo Xu, Dr. Zhibin Ning, Dr. Janice Mayne, Dr. Kerwin Chiang, Dr. Rui Chen, Dr. Xu Zhang, and Jasmine Moore for all the motivation, scientific discussion, and friendship over the course of my two years in the lab. I would also like to thank Ngoc Vuong for his friendship and support during my undergraduate thesis at Health Canada, and the mentorship of Dr. Renaud Vincent which has inspired me to pursue the path of scientific research. Finally, I would like to thank my friends, family and Matt. Their support and encouragement helped me get through the joys and the hardships both in life and during the course of my studies. iii STATEMENT OF CONTRIBUTION All experiments were conducted by Alexandra Star (AS). Experimental design was conducted by Daniel Figeys (DF), Janice Mayne (JM) and Zhibin Ning (ZN) and AS. Data analysis was conducted by AS with the advice and mentoring of ZN. iv TABLE OF CONTENTS ABSTRACT .......................................................................................................................................... ii ACKNOWLEDGEMENTS .................................................................................................................. iii STATEMENT OF CONTRIBUTION ................................................................................................. iv ABBREVIATIONS ............................................................................................................................ viii LIST OF FIGURES .............................................................................................................................. xi LIST OF TABLES ............................................................................................................................... xii LIST OF APPENDICES ..................................................................................................................... xii 1. INTRODUCTION ............................................................................................................................. 1 I. Proteomics and Post-Translational Modifications.......................................................................... 1 II. Protein Methylation ...................................................................................................................... 2 i. Arginine Methylation ................................................................................................................. 3 ii. Lysine Methylation .................................................................................................................... 4 III. Biological Roles of Protein Methylation ..................................................................................... 7 i. Protein Methylation in Transcriptional Regulation .................................................................... 7 ii. Methylation in mRNA splicing ................................................................................................. 9 iii. Effect of Methylation on Protein Stability ............................................................................. 10 IV. Protein Methylation in Disease ................................................................................................. 11 i. Cancer ....................................................................................................................................... 11 ii. Embryo Development .............................................................................................................. 12 iii. Methylation in Vaccination .................................................................................................... 13 iv. Cardiovascular Disease ........................................................................................................... 14 v. Spinal Muscular Atrophy ......................................................................................................... 14 V. Current Approaches to Studying Methylation ............................................................................ 15 i. Low-Throughput Targeted Approaches ................................................................................... 15 ii. High-Throughput Enrichment-Based Screening ..................................................................... 15 iii. Methyl-Lysine Binding Domains to Enrich for Methylated Proteins .................................... 16 iv. Pan-Methyl Antibodies to Enrich for Methylated Peptides .................................................... 16 v. Derivatization Coupled with Affinity Enrichment .................................................................. 17 vi. Click Chemistry to Determine Methyltransferase Targets ..................................................... 17 vii. Predictive Modeling of Methylation Sites ............................................................................. 18 VI. Novel Approach to Enrich for Methylated Peptides ................................................................. 18 VII. Rationale .................................................................................................................................. 20 v VIII. Statement of Hypothesis and Objectives ................................................................................ 21 i. Hypothesis ................................................................................................................................ 21 ii. Objectives ................................................................................................................................ 21 2. MATERIALS AND METHODS .................................................................................................... 26 I. Cell Culture .................................................................................................................................. 26 II. Heavy-Methionine Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) ............ 26 III. Cell Lysis ................................................................................................................................... 27 IV. Subcellular Fractionation........................................................................................................... 27 V. Immunoblotting .......................................................................................................................... 28 VI. Proteomics Sample Preparation ................................................................................................. 29 i. Protein Precipitation ................................................................................................................. 29 ii. In-solution Protein Digestion .................................................................................................