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Global Proteomic Detection of Native, Stable, Soluble Human Protein Complexes

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Global Proteomic Detection of Native, Stable, Soluble Human Protein Complexes GLOBAL PROTEOMIC DETECTION OF NATIVE, STABLE, SOLUBLE HUMAN PROTEIN COMPLEXES by Pierre Claver Havugimana A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto © Copyright by Pierre Claver Havugimana 2012 Global Proteomic Detection of Native, Stable, Soluble Human Protein Complexes Pierre Claver Havugimana Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto 2012 Abstract Protein complexes are critical to virtually every biological process performed by living organisms. The cellular “interactome”, or set of physical protein-protein interactions, is of particular interest, but no comprehensive study of human multi-protein complexes has yet been reported. In this Thesis, I describe the development of a novel high-throughput profiling method, which I term Fractionomic Profiling-Mass Spectrometry (or FP-MS), in which biochemical fractionation using non-denaturing high performance liquid chromatography (HPLC), as an alternative to affinity purification (e.g. TAP tagging) or immuno-precipitation, is coupled with tandem mass spectrometry-based protein identification for the global detection of stably- associated protein complexes in mammalian cells or tissues. Using a cell culture model system, I document proof-of-principle experiments confirming the suitability of this method for monitoring large numbers of soluble, stable protein complexes from either crude protein extracts or enriched sub-cellular compartments. Next, I document how, using orthogonal functional genomics information generated in collaboration with computational biology groups as filters, we applied FP-MS co-fractionation profiling to construct a high-quality map of 622 predicted unique soluble human protein complexes that could be biochemically enriched from HeLa and HEK293 nuclear and cytoplasmic extracts. Our network is enriched in assemblies consisting of human disease-linked proteins and contains hundreds of putative new components and novel ii complexes, many of which are broadly evolutionarily conserved. This study revealed unexpected biological associations, such as the GNL3, FTSJ3, and MKI67IP factors involved in 60S ribosome assembly. It is my expectation that this first systematic, experimentally-derived atlas of putative human protein complexes will constitute a starting point for more in depth, hypothesis- driven functional investigations of basic human molecular and cellular biology. I also note that my generic FP-MS screening approach can, and is currently, being applied by other members of the Emili laboratory to examine the global interactomes of other mammalian cell lines, tissues, sub-cellular compartments, and diverse model organisms, which should expand our understanding of proteome adaptations and association networks associated with cell physiology, animal development and molecular evolution. iii Acknowledgments I thank God for protecting and guiding me throughout the most hopeless situations of my life and allowing me to arise as a mature, responsible, stronger and wiser individual ready to face any challenge with faith and perseverance. From what began as just gaining a diploma in order to establish myself in my new country and home after the genocide in Rwanda, later ignited a dream which led to a lengthy and incredible journey of doctoral studies. I would like to thank several people who have helped me to make this journey a very rewarding and memorable experience. First of all, I would like to express my sincere appreciation and gratitude to my supervisor, Dr. Andrew Emili. I joined the Emili laboratory in 2005, first as a Research Assistant and then later as a Graduate Student starting 2007, after obtaining degrees in Biochemistry and Chemistry and a Master of Science in Chemical Engineering, all from Laval University. Along this path, Dr. Andrew Emili has provided me the resources and freedom to test my every single idea, as well as tremendous encouragement and unwavering support, all with outstanding patience. I consider Dr. Emili as my godparent, a colleague and a great friend with whom I received invaluable advice that has and will always remind me of my parents to whom I inherited the value of the hard work and perseverance. Second, I would like to thank all members of my PhD exam committee for their valuable time and constructive criticisms. I particular thank my supervisory committee members, Dr. Gary Bader, Dr. Jack Greenblatt and Dr. Andrew Wilde, for educating me and allowing me to grow as a critical thinking scientist. Their encouragement and guidance through challenge in the past five years of my doctoral studies have been a source of inspiration and I hope I can live up to their expectations. iv Third, I respectfully acknowledge my exceptional collaborators, the group of Dr. Edward Marcotte, Dr. Alberto Paccanaro, Dr. Shoshana Wodak and Dr. Elisabeth Tillier. Their computational expertise and intellectual inputs made the findings in this thesis a dream that came true. Special thanks to Dr. Edward Marcotte for the opportunity afforded me to work in his laboratory, and to members of his team for their support and friendship during my stay in Austin. Thanks are also expressed to Dr. Jim Ingles whose comments and editing rules made an everlasting improvement to this thesis. Fourth, I also greatly appreciated the support, advice and encouragement from the present and past members of the Emili and Greenblatt labs during our daily conversations, lab meetings and exam preparation. In particular, I would like to extend my sincere thanks to Vincent Fong, Pingzhao Hu, Sadhna Phanse, Cuihong Wan, Hongbo Guo and Zuyao Ni for their technical assistance, to Ruth Isserlin, Gabe Musso and Jeffrey Fillingham for proofreading my committee reports, and to Alla Gagarinova for proofreading my re-class proposal. Fifth, I would like to thank my wife and best friend, Venis, for taking pride in my achievements. Your patience and unprecedented support and belief in my personal goals and potential has meant so much to me. Thanks are also due to my cousin, Ignace and his family, and my sister, Donathille, for being there when I needed them most. A huge thanks to all members of my family-in-law for the warmth and love they have brought into my life. Finally, I dedicated this thesis, the most significant achievement of my life so far, to all members of my family who could have been proud of me today if their lives were not taken so early in the 1994 Rwandan genocide. Thank you all and God bless you. Pierre Claver Havugimana, 2012 v Table of Contents Acknowledgments .......................................................................................................................... iv Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................. x List of Abbreviations .................................................................................................................... xii Chapter 1 Introduction and Background Information ..................................................................... 1 1 Introduction and background information ................................................................................. 1 1.1 The importance of protein complexes ................................................................................. 1 1.2 Experimental approaches for genome-wide screening of protein complexes .................... 5 1.2.1 Affinity purification and mass spectrometry .......................................................... 9 1.2.2 Low-throughput experimental approaches amenable to proteome scale-up ......... 26 1.3 Computational methods for prediction and analysis of protein complexes ...................... 29 1.4 Repositories of experimentally derived PPI and protein complex data ............................ 32 1.5 Goal and purposes of the present thesis project ................................................................ 33 Chapter 2 Development of a High-Throughput Global Profiling Method Based on High Resolution Ion-Exchange High Performance Liquid Chromatography for Proteomic-Scale Analyses of Native Stable Soluble Protein Complexes ........................................................... 35 2 Development of a high-throughput global profiling method based on high resolution ion- exchange high performance liquid chromatography for proteomic-scale analyses of native stable soluble protein complexes ............................................................................................. 36 2.1 Introduction ....................................................................................................................... 36 2.2 Material and Methods ....................................................................................................... 39 2.2.1 Cell lines and cell-free extract preparation ........................................................... 39 2.2.2 HPLC columns, buffers, and instrumentation ....................................................... 39 2.2.3 Single phase weak cation-exchange fractionation of HeLa cytosolic extracts ..... 39 2.2.4 Single phase weak anion-exchange
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