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Developing Next Generation Technologies for Spatially Targeted Developing Next Generation Technologies for Spatially Targeted Proteomics By Daniel Joseph Ryan 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 June 30th, 2019 Nashville, Tennessee Approved: Richard M. Caprioli, Ph.D. Jeffrey M. Spraggins, Ph.D. John A. McLean, Ph.D. Lars Plate, Ph.D. Kevin L. Schey, Ph.D. Copyright © 2019 by Daniel Joseph Ryan All Rights Reserved ii ACKNOWLEDGEMENTS It is with the help of many people that I am afforded the unique privilege of being able to sit here and write out an acknowledgements section for my dissertation. First and foremost, I would like to thank both of advisors, Dr. Richard Caprioli and Dr. Jeff Spraggins. Richard, you have pushed me both scientifically and personally. You have led by example and I am very grateful to have had the opportunity to spend my graduate career in your laboratory, it is not something I take for granted. Jeff, you helped me gain traction upon entering the lab, gave me direction, and have been an integral part of my journey while at Vanderbilt. You went above and beyond what is expected of any advisor to help mold me into the scientist I am today, and I am grateful to call you a mentor and more importantly, a friend. To my entire committee, Kevin Schey, John McLean, and Lars Plate; I am forever thankful for the time you have taken to help push me towards excellence throughout this journey. I want to thank my lab mates, who are also my closest friends, for their support and friendship throughout this period of my life. Thank you for dealing with my craziness and also for being as excited for Happy Hour as I am, I couldn’t imagine going through this process with another group of people. I will always hold the many memories involving late night Patriots at Dukes, SatCo porch beers, and appropriately priced snacks at Answer close to my heart, and consider them a contributing factor in finishing my research at Vanderbilt. To the staff of the MSRC, you all have been instrumental in ensuring I stay happy and on track, and I am so thankful. Maureen Casey, you were truly one of the best parts of coming to lab every day, and I wish you the best in retirement. iii Finally, and most importantly, I would like to thank my family and my daughter for their continued support. There is an old quote that states, “To us, family means putting your arms around each other and being there”, and I have been truly blessed with a family, with parents, who have never swayed from that motto. Only they know how truly difficult this path has been at times, and they have come to my aid whenever I needed it, and I am truly thankful for all that you guys have done. To my beautiful, smart, and funny daughter Norah, you have no idea how important your presence, whether near or far, served as the perfect distraction to all of the fuss of chemistry and graduate school. Walking to daycare after lab each day, picking up an always surprised 2 year old, or watching you shovel loads of hummus in your mouth on my couch, to nightly runs in a jogging stroller. These are some of my fondest memories in Nashville. The research detailed below is a culmination of not only my work, but our combined work as a family, and any success I have in the future, and have had in the past, is a direct result of all of you. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ......................................................................................................................... iii LIST OF FIGURES ................................................................................................................................... viii LIST OF TABLES ....................................................................................................................................... xi LIST OF ABBREVIATIONS ..................................................................................................................... xii Chapter I. MATRIX-ASSISTED LASER DESORPTION/IONIZATION IMAGING MASS SPECTROMETRY AND PROTEIN IDENTIFICATION Overview .................................................................................................................................................. 1 Introduction .............................................................................................................................................. 2 Matrix-Assisted Laser Desorption/Ionization and Mass Spectrometry ................................................... 2 MALDI Imaging Mass Spectrometry and MALDI Profiling of Proteins from Tissue ............................ 4 Time-of-Flight Mass Spectrometry .......................................................................................................... 8 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry ........................................................ 10 Protein Identification in MALDI IMS .................................................................................................. 13 Protein Identification Strategies ............................................................................................................. 16 Tissue Homogenization ...................................................................................................................... 17 In Situ Enzymatic Digestion ................................................................................................................ 18 Chemical Derivatization ..................................................................................................................... 19 Laser Capture Microdissection .......................................................................................................... 20 Hydrogel Extraction ........................................................................................................................... 20 Liquid Extraction Surface Analysis .................................................................................................... 21 Research Summary and Objectives ........................................................................................................ 23 II. IMPROVING PROTEIN ION TRANSMISSION ON A 15T FT-ICR MS FOR MALDI IMS Overview ................................................................................................................................................ 25 Introduction ............................................................................................................................................ 25 Results .................................................................................................................................................... 28 Systematic Study of Source Pressure .................................................................................................. 28 Tissue Imaging and Image Fusion ...................................................................................................... 37 Discussion .............................................................................................................................................. 45 Methods.................................................................................................................................................. 46 Mass Spectrometry .............................................................................................................................. 46 v Imaging Mass Spectrometry ................................................................................................................ 48 Data Driven Multi-Modal Image Fusion ............................................................................................ 49 III. SPATIALLY TARGETED PROTEOMIC ANALYSIS FROM TISSUE USING LIQUID EXTRACTION SURFACE ANALYSIS Overview ................................................................................................................................................ 51 Introduction ............................................................................................................................................ 51 Results .................................................................................................................................................... 55 Surface Extraction Performance .......................................................................................................... 55 Correlating IMS with LESAplusLC Protein Experiments .................................................................. 62 Discussion .............................................................................................................................................. 72 Methods.................................................................................................................................................. 73 Tissue Preparation .............................................................................................................................. 73 MALDI IMS ......................................................................................................................................... 74 In Situ Tryptic Digestion ..................................................................................................................... 75 Tissue Extractions ................................................................................................................................ 75 Online LC-MS ....................................................................................................................................
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