ABSTRACT Title of Thesis: INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS Katherine R. Adams, Master of Science, 2016 Thesis Directed By: Catherine Fenselau, Professor, Department of Chemistry and Biochemistry Exosomes released by myeloid-derived suppressor cells (MDSC) are 30 nm in diameter extracellular vesicles that have been shown to carry biologically active proteins as well as ubiquitin molecules. Ubiquitin is known to have many functions, including involvement in the formation of exosomes, although the exact role is highly contested. In the study reported here, the proteome and ubiquitome of MDSC exosomes has been investigated by bottom-up proteomics techniques. This report identifies more than 1000 proteins contained in the MDSC exosome cargo and 489 sites of ubiquitination in more than 300 ubiquitinated proteins based on recognition of glycinylglycine tagged peptides without antibody enrichment. This has allowed extensive chemical and biological characterization of the ubiquitinated cohort compared to that of the entire protein cargo to support hypotheses on the role of ubiquitin in exosomes. INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS by Katherine R. Adams Thesis submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Master of Science 2016 Advisory Committee: Professor Catherine Fenselau, Chair Professor Neil Blough Professor Nicole LaRonde © Copyright by Katherine R. Adams 2016 Dedication This work is dedicated to my parents, David and Mary Lou Adams, who have always supported me in all walks of life and taught me the value of hard work. To my older brother Matthew who taught me all of life’s important lessons. And to my boyfriend and best friend Adam who was always available for a pep talk and food. ii Acknowledgements First and foremost I would like to acknowledge my advisor Dr. Catherine Fenselau. It has been such an honor to work in this research group these past two years and learn as much as I could. Thank you for your guidance and invaluable wisdom that I will carry with me throughout my career. I would like to acknowledge my collaborators who have made this project possible. I would like to thank Dr. Suzanne Ostrand-Rosenberg and Virginia Clements for providing the MDSC exosomes and for help with understanding the cell biology. I would also like to thank Dr. Yan Wang for help and guidance using the orbitrap Fusion Lumos, an amazing and incredibly expensive instrument that has been a highlight of my graduate school work. I would like to thank Dr. Nathan Edwards for always being available to answer bioinformatics questions. I would also like to thank my committee members Dr. Neil Blough and Dr. Nicole LaRonde. I would like to thank all past and current members of the Fenselau group including Lucia Geis Asteggiante, Sitara Chauhan, Yeji Kim, Mrs. Sara Moran, Dr. Dapeng Chen, Dr. Amanda Lee, and Dr. Meghan Burke. I would also like to thank the amazing group of graduate students I have met while at Maryland; my gal pals Leila Duman, Teodora Kljaic, Sarah Robinson, and Emily Sahadeo, my crazy fun roommates Noah Masika, Kyle Oliver, and Vincent Wu, and an extra special thank you to my soul friend Samantha Nowak who I will never stop being thankful for. Lastly I would like to thank my family for their unconditional love and support. iii Table of Contents Dedication ..................................................................................................................... ii Acknowledgements ...................................................................................................... iii Table of Contents ......................................................................................................... iv List of Tables ................................................................................................................ v List of Figures .............................................................................................................. vi List of Abbreviations ................................................................................................. viii Chapter 1: Introduction ................................................................................................. 1 1.1 Proteomics........................................................................................................... 1 1.1.1 Overview ...................................................................................................... 1 1.1.2 Orbitrap Fusion Lumos Tribrid .................................................................... 5 1.2 Biological question ............................................................................................. 7 1.2.1 Exosome biogenesis and function ................................................................ 7 1.2.2 Ubiquitin as a post-translational modification ............................................. 8 1.2.3 The role of ubiquitin in exosome formation .............................................. 11 1.3 Research objectives and significance ................................................................ 12 Chapter 2: Identification of proteins in the exosome lysate ....................................... 14 2.1 Introduction ....................................................................................................... 14 2.2 Materials and methods ...................................................................................... 16 2.2.1 Materials .................................................................................................... 16 2.2.2 Biological sample preparation ................................................................... 16 2.2.3 Protein analysis .......................................................................................... 16 2.2.4 LC-MS/MS and bioinformatics ................................................................. 17 2.3 Results and discussion ...................................................................................... 19 2.4 Conclusions ....................................................................................................... 27 Chapter 3: The Ubiquitome ........................................................................................ 29 3.1 Introduction ....................................................................................................... 29 3.2 Materials and methods ...................................................................................... 32 3.2.1 Materials .................................................................................................... 32 3.2.2 Biological sample preparation and protein analysis .................................. 32 3.2.3 Immunoprecipitations ................................................................................ 33 3.2.4 Western blotting ......................................................................................... 34 3.3 Results and discussion ...................................................................................... 34 3.3.1 Confirmation of ubiquitinated proteins by immunoprecipitation .............. 34 3.3.2 Bottom-up identification of ubiquitinated proteins.................................... 36 3.4 Conclusions ....................................................................................................... 42 Appendices .................................................................................................................. 45 Bibliography ............................................................................................................... 86 iv List of Tables Table 2.1 – Some biologically active proteins identified in the exosome lysate. Table 2.2 – Proteasome components identified in the exosome lysate. Table 2.3 – Ubiquitination pathway enzymes identified in the exosome lysate. v List of Figures Figure 1.1. Diagram of the fragmentation and ion types that can occur along the peptide backbone. Figure 1.2. Schematic of the orbitrap Fusion Lumos Tribrid mass spectrometer. (http://planetorbitrap.com/) Figure 1.3. Illustration of the formation of multi-vesicular bodies and intraluminal vesicles, and their pathway to degradation in the lysosome or release as exosomes. Figure 1.4. An overview of the components involved in the ubiquitin system in mammalian cells. Figure 2.1. A flowchart summarizing sample preparation and this bottom-up workflow. Figure 2.2a-b. GO annotations of the (a) molecular functions and (b) cellular components for the whole exosome lysate. Figure 2.3a-b. (a) Isoelectric point, pI, distribution of the proteins identified in the exosome lysate and (b) a comparison of the isoelectric point distributions of the exosome lysate and mouse proteome (available from http://isoelectricpointdb.org/index.html). Figure 2.4. MW distributions of the proteins identified in the MDSC exosomes; inset shows the 0 - 350,000 Da range. Figure 3.1. A bottom-up proteomic workflow for ubiquitinated proteins utilizing digestion by trypsin and activation by CID. (Adapted from references 11 and 39). vi Figure 3.2a-f. Western blots probing with anti-ubiquitin antibody #3933 of (a) exosome lysate, (b) eluate of #3933 immunoprecipitation, (c) non-bound fraction from #3933 immunoprecipitation, (d) negative control of #3933 immunoprecipitation, (e) eluate