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I HIGH MASS ACCURACY COUPLED to SPATIALLY-DIRECTED HIGH MASS ACCURACY COUPLED TO SPATIALLY-DIRECTED PROTEOMICS FOR IMPROVED PROTEIN IDENTIFICATIONS IN IMAGING MASS SPECTROMETRY EXPERIMENTS By David Geoffrey Rizzo Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Chemistry August, 2016 Nashville, Tennessee Approved: Richard M. Caprioli, Ph.D. Kevin L. Schey, Ph.D. John A. McLean, Ph.D. Michael P. Stone, Ph.D. i Copyright © 2016 by David Geoffrey Rizzo All Rights Reserved ii This work is dedicated to my family and friends, who have shown nothing but support for me in all of life’s endeavors. iii ACKNOWLEDGEMENTS “As we express our gratitude, we must never forget that the highest appreciation is not to utter words, but to live by them.” - John F. Kennedy – There are many people I must thank for showing kindness, encouragement, and support for me during my tenure as a graduate student. First and foremost, I would like to thank my research advisor, Richard Caprioli, for providing both ample resources and guidance that allowed me to grow as a scientist. Our discussions about my research and science in general have helped me become a much more focused and discerning analytical chemist. I must also thank my Ph.D. committee members, Drs. Kevin Schey, John McLean, and Michael Stone, who have brought valuable insight into my research and provided direction along the way. My undergraduate advisor, Dr. Facundo Fernández, encouraged me to begin research in his lab and introduced me to the world of mass spectrometry. It was his motivation that drove me to pursue my Ph.D. Every member of the Mass Spectrometry Research Center, both past and present, has guided me in some form or fashion. I would not be where I am today without the support of my “lab family”, both on a personal and scientific level. Maureen Casey was always there to brighten up the lab with humor and support. I will certainly miss going to see “Mama Maureen” and have her fix any problem almost immediately. Dr. Peggi Angel advised me as a rotation student when I first came to Vanderbilt and I strived to emulate her passion for science. Amanda Hachey, Salisha Hill, and Dr. Kristie Rose in the Proteomics Core could not have been more accommodating as I plunged into the vast field of proteomics. They were always there to share their wisdom, helping me learn new techniques that will be invaluable in my career. Drs. Megan iv Gessel, Joshua Nicklay, Glenn Harris, Jeffrey Spraggins, and Boone Prentice have all been vital to my development as a scientist, acting as soundboards for ideas and encouraging me to enjoy successes and learn from failures. Whenever I needed to vent about science or life, they were there to listen. The fellow graduate students in lab who have seen me through this journey have left a lasting impression on me as well. They have always been there to lend a hand and I will truly miss seeing them in lab every day. None of this would be possible without support from my family and friends. My mother, Robin, and father, Joe, encouraged me to pursue my love of science from an early age, even if that meant driving to enrichment programs early Saturday mornings. They taught me how to be a hard worker and independent person, but to never forget that no man is an island. Success and humility should never be mutually exclusive. My brother, Joey, and my sister, Meghan, continue to encourage me every day in everything that I do. Meghan has listened to the recap of my current state of life at day’s end, and for that, I am eternally grateful. I cannot forget Rebecca Rosenberg, who has been a part of our family since the first grade. We have grown up together and will always be there to ground each other, no matter how far apart we are. My dogs, Marco and Polo, are the first things I see when I wake up and the last things I see when I go to bed. They’ve made the roughest days better, always showing excitement and affection when I get home (no matter how late that may be). Lastly, I would like to thank my entire extended family, including all of my aunts, uncles, and cousins. Though my grandparents could not be here to see this achievement, I know they would all be proud of such an accomplishment. I must also acknowledge the funding support of grants from the NIH/NIGMS (5P41 GM103391-05 and 5R01 GM058008), the NIH Shared Instrumentation Grant Program (1S10OD012359-01), and a fellowship from Aegis Sciences Corporation. v TABLE OF CONTENTS Page DEDICATION ............................................................................................................................... iii ACKNOWLEDGEMENTS ........................................................................................................... iv TECHNICAL ABBREVIATIONS ................................................................................................ ix LIST OF TABLES ......................................................................................................................... xi LIST OF FIGURES ..................................................................................................................... xiii Chapter I. INTRODUCTION & RESEARCH OBJECTIVES ...................................................................1 Matrix-Assisted Laser/Desorption Ionization Mass Spectrometry ..........................................1 Imaging Mass Spectrometry .....................................................................................................3 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry ...........................................5 Spatially-Directed Tissue Collection Methods .........................................................................9 Traditional Approaches to Protein Identification in IMS Experiments ..................................11 Summary and Research Objectives ........................................................................................14 II. MALDI FTICR IMS OF INTACT PROTEINS: USING MASS ACCURACY TO LINK PROTEIN IMAGES WITH PROTEOMICS DATA..............................................16 Introduction ............................................................................................................................16 Methodologies for MALDI FTICR IMS and Identification of Intact Proteins ......................17 Tissue Preparation ..............................................................................................................17 Matrix Application .............................................................................................................17 MALDI FTICR IMS ..........................................................................................................18 Histology ............................................................................................................................18 Protein Purification for Identifications ..............................................................................19 LC-Coupled Tandem Mass Spectrometry (ETD) ..............................................................19 Data Analysis .....................................................................................................................20 MALDI FTICR IMS of Rat Brain as a Model System for Protein Imaging ..........................21 Observed Protein Expression in clear cell Renal Cell Carcinoma (ccRCC) ..........................36 Conclusions ............................................................................................................................39 III. SPATIALLY-TARGETED EXTRACTIONS TO IMPROVE PEPTIDE IDENTIFICATIONS FROM MALDI FTICR IMS EXPERIMENTS ....................................40 Introduction ............................................................................................................................40 vi Methodologies for Trypsin Application, in situ Digestion, Incubation and MALDI IMS for Peptides .......................................................................................................41 Tissue Preparation ..............................................................................................................41 Trypsin Digestion...............................................................................................................42 Matrix Application .............................................................................................................42 MALDI FTICR IMS .........................................................................................................43 Peptide Extraction from Tissue ..........................................................................................43 LC-Coupled Tandem Mass Spectrometry (HCD) .............................................................43 Data Analysis .....................................................................................................................44 Optimizing Incubation Conditions to Maximize Digestion and Minimize Delocalization .........................................................................................................................44 MALDI FTICR IMS and Localized Peptide Analysis of Biological Samples ......................48 Conclusions ............................................................................................................................57 IV. ADVANCED HYDROGEL TECHNOLOGIES FOR ENHANCED ON-TISSUE PROTEIN DIGESTION AND IMPROVED SPATIAL LOCALIZATION ...........................58 Introduction ............................................................................................................................58
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