Development and Application of Mass Spectrometry-Based Proteomics to Generate and Navigate the Proteomes of the Genus Populus
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University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2013 DEVELOPMENT AND APPLICATION OF MASS SPECTROMETRY- BASED PROTEOMICS TO GENERATE AND NAVIGATE THE PROTEOMES OF THE GENUS POPULUS Paul Edward Abraham [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Biochemistry Commons, Biology Commons, Cell Biology Commons, Forest Biology Commons, Genomics Commons, Molecular Biology Commons, Plant Biology Commons, and the Systems Biology Commons Recommended Citation Abraham, Paul Edward, "DEVELOPMENT AND APPLICATION OF MASS SPECTROMETRY-BASED PROTEOMICS TO GENERATE AND NAVIGATE THE PROTEOMES OF THE GENUS POPULUS. " PhD diss., University of Tennessee, 2013. https://trace.tennessee.edu/utk_graddiss/1692 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Paul Edward Abraham entitled "DEVELOPMENT AND APPLICATION OF MASS SPECTROMETRY-BASED PROTEOMICS TO GENERATE AND NAVIGATE THE PROTEOMES OF THE GENUS POPULUS." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Life Sciences. Robert Hettich, Major Professor We have read this dissertation and recommend its acceptance: Mircea Podar, Steven Wilhelm, Albrecht von Arnim, Loren Hauser Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) DEVELOPMENT AND APPLICATION OF MASS SPECTROMETRY-BASED PROTEOMICS TO GENERATE AND NAVIGATE THE PROTEOMES OF THE GENUS POPULUS A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Paul Edward Abraham May 2013 ACKNOWLEDGEMENTS First, I’d like to gratefully and sincerely thank Dr. Robert Hettich for guiding me through the completion of my dissertation and subsequent Ph.D. Bob’s open door policy, relaxed demeanor, and constant encouragement made for a good working relationship. His mentorship was paramount in providing a well-rounded graduate school experience. I am grateful for all of the opportunities he has provided, especially all of those “vacations” at the annual American Society for Mass Spectrometry conferences. Over these past 7 years at Oak Ridge National Laboratory (two of those spent as an undergraduate research assistant), I’ve learned so much from Bob and I could not thank him enough. If imitation is the sincerest form of flattery and gratitude, I’d like to highlight a few of his colorful ways of giving advice (a.k.a. Bobisms): team work (“We are all pegs on a stool”, “We are all in the same boat and we shoot the swimmers”, and “A rising tide lifts all boats”), life’s complications (“A fly in the ointment”, “Life is a complicated landscape”, “It’s all dynamic”, and “It’s a moving target”, “Tipping your hand”, “Chopping at the bit”, and “Raise the red herring up the flagpole”), prioritization (“Too many irons in the fire, “Your dance card is full”, “What’s on the docket”, and “We are going to build a white fence so that if you reach outside, you get your arm chopped off”), and a manuscript in progress (“Put all your eggs in a basket and shoot for the homerun fence”, “Don’t throw the baby out with the bathwater”, “First show me the baby and then you can tell me about the labor pains”, “The onus is on us”, “An albatross around our necks”, and “From soup to nuts”). Secondly, I’m very grateful to the remaining members of my dissertation committee, Dr. Albrecht von Arnim, Dr. Loren Hauser, Dr. Mircea Podar, and Dr. Steven Wilhelm. Their academic support and input are greatly appreciated. I’d also like to express my gratitude to Dr. Gerald Tuskan for his mentorship, guidance and support. My gratitude is also extended to other members of Bob’s research group. Specifically, I’d like to thank Dr. Nathan Verberkmoes, Dr. Richard Giannone, Dr. Brian Erickson, Dr. Alison Erickson, Dr. Andrew Dykstra, and Dr. Rachel Adams for their guidance, discussions and suggestions throughout my graduate career. Thirdly, I would like to thank my parents Paul and Cindy Abraham. They have both instilled many qualities in me, giving me a foundation with which to live honestly, responsibly, and compassionately. I would also like to thank my brothers Andy and Ryan Abraham, my little sister Leslie Abraham, as well as my mother-in-law Terry Lynn Hursey. I would also like to thank the three best friends that anyone could have, Jason Stewart, Brandon Barger, and Dr. Andy Harris. All of you helped shape me into the person I am today. Thank you. Finally, and most importantly, my upmost gratitude goes to my beautiful wife Annie. Her encouragement, support, patience and constant selflessness were undeniably the foundation upon which the past four years of my life have been built. Although she endured many, many long hours alone while I worked on my dissertation, I was always greeted with a smile. Last, but certainly not least, I must acknowledge my son Parker Edward Abraham. At only 4 months old, you inspired me to never surrender the goals I have set. ii ABSTRACT Historically, there has been tremendous synergy between biology and analytical technology, such that one drives the development of the other. Over the past two decades, their interrelatedness has catalyzed entirely new experimental approaches and unlocked new types of biological questions, as exemplified by the advancements of the field of mass spectrometry (MS)-based proteomics. MS-based proteomics, which provides a more complete measurement of all the proteins in a cell, has revolutionized a variety of scientific fields, ranging from characterizing proteins expressed by a microorganism to tracking cancer-related biomarkers. Though MS technology has advanced significantly, the analysis of complicated proteomes, such as plants or humans, remains challenging because of the incongruity between the complexity of the biological samples and the analytical techniques available. In this dissertation, analytical methods utilizing state-of- the-art MS instrumentation have been developed to address challenges associated with both qualitative and quantitative characterization of eukaryotic organisms. In particular, these efforts focus on characterizing Populus, a model organism and potential feedstock for bioenergy. The effectiveness of pre-existing MS techniques, initially developed to identify proteins reliably in microbial proteomes, were tested to define the boundaries and characterize the landscape of functional genome expression in Populus. Although these approaches were generally successful, achieving maximal proteome coverage was still limited by a number of factors, including genome complexity, the dynamic range of protein identification, and the abundance of protein variants. To overcome these challenges, improvements were needed in sample preparation, MS instrumentation, and bioinformatics. Optimization of experimental procedures and implementation of current state-of-the-art instrumentation afforded the most detailed look into the predicted proteome space of Populus, offering varying proteome perspectives: 1) network-wide, 2) pathway-specific, and 3) protein-level viewpoints. In addition, we implemented two bioinformatic approaches that were capable of decoding the plasticity of the Populus proteome, facilitating the identification of single amino acid polymorphisms and generating a more accurate profile of protein expression. Though the methods and results presented in this dissertation have direct implications in the study of bioenergy research, more broadly this dissertation focuses on developing techniques to contend with the notorious challenges associated with protein characterization in all eukaryotic organisms. iii TABLE OF CONTENTS CHAPTER 1 ....................................................................................................................... 1 Principles of Mass Spectrometry-based Proteomics and its Applications to a Bioenergy Crop..................................................................................................................................... 1 1.1 The Omics Era: Enabling a Systems-level View of How Cells Function ................ 1 1.2 Mass Spectrometry-based Proteomics ...................................................................... 4 1.3 Bottom-up Proteomics for Discovery-based Investigations ..................................... 8 1.3.1 Peptide Sequencing by Tandem Mass Spectrometry ......................................... 8 1.3.2 Peptide Identification by Searching Algorithms .............................................. 12 1.3.3 Protein Inference .............................................................................................. 16 1.3.4 Protein Quantitation ......................................................................................... 18 1.4 Proteomics of a Bioenergy-relevant Organism ....................................................... 22 1.4.1 Introduction to Bioenergy ................................................................................ 22