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UCLA Electronic Theses and Dissertations UCLA UCLA Electronic Theses and Dissertations Title A Liquid Chromatography-Mass Spectrometry Platform for Identifying Protein Targets of Small-Molecule Binding Relevant to Disease and Metabolism Permalink https://escholarship.org/uc/item/8pn170t7 Author O'Brien Johnson, Reid Publication Date 2018 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA Los Angeles A Liquid Chromatography-Mass Spectrometry Platform for Identifying Protein Targets of Small-Molecule Binding Relevant to Disease and Metabolism A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biochemistry and Molecular Biology by Reid Lee O’Brien Johnson 2018 ABSTRACT OF THE DISSERTATION A Liquid Chromatography-Mass Spectrometry Platform for Identifying Protein Targets of Small-Molecule Binding Relevant to Disease and Metabolism by Reid Lee O’Brien Johnson Doctor of Philosophy in Biochemistry and Molecular Biology University of California, Los Angeles, 2018 Professor Joseph Ambrose Loo, Chair Identifying small-molecule binders to protein targets remains a daunting task due to the huge diversity in compound structure, activity, and mechanisms of action. Affinity- based target identification techniques are limited by the necessity to modify each drug individually (without losing bioactivity), while non-affinity based approaches are dependent on the drug’s ability to induce specific biochemical/cellular readouts. To overcome these limitations, we have developed a high-throughput liquid chromatography- mass spectrometry (LC-MS) platform coupled to a universally applicable target identification approach that analyzes direct small-molecule binding to its protein target(s). DARTS (drug affinity responsive target stability) relies on a well-known phenomenon in which ligand binding causes thermodynamic stabilization of its target protein’s structure such that the protein becomes resistant to a variety of insults, including proteolysis. ii DARTS-MS is performed by screening for proteins that become resistant to proteolysis in the presence of the ligand. Protection from proteolysis is specific to the target protein(s) while proteolysis of non-target proteins is unchanged, allowing for unbiased, proteome-wide screening for small-molecule binders. Samples are proteolyzed and fractionated according to molecular weight (MW). Each molecular weight range is subsequently analyzed by LC-MS/MS (Q-Exactive q-Orbitrap) to identify and quantify the proteins present. Proteins that are differentially digested between a small-molecule- protected sample and the control are determined using Protomap (Protein Topography and Migration Analysis Platform) based on differences in the total amount of each protein and/or differences in sizes of the protein fragments. To date we have utilized our DARTS- MS strategy both to re-discover known protein targets of existing small-molecules as well as discovering novel targets for established drugs and important metabolites. iii This dissertation of Reid Lee O’Brien Johnson is approved. James Akira Wohlschlegel Robert Thompson Clubb Joseph Ambrose Loo, Committee Chair University of California, Los Angeles 2018 iv DEDICATION I would like to dedicate this work to all my friends and family for their love and support over the years. v TABLE OF CONTENTS ABSTRACT OF THE DISSERTATION .............................................................................ii DEDICATION .................................................................................................................. v TABLE OF CONTENTS ..................................................................................................vi LIST OF FIGURES ........................................................................................................ viii LIST OF TABLES ............................................................................................................ x ACKNOWLEDGMENTS ..................................................................................................xi VITA .............................................................................................................................. xiii CHAPTER 1 : CURRENT LIMITATIONS IN IDENTIFYING PROTEIN TARGETS OF SMALL MOLECULES ..................................................................................................... 1 1.1 INTRODUCTION ................................................................................................ 1 1.2 CURRENT APPROACHES ................................................................................ 4 1.3 DARTS DEVELOPMENT ................................................................................... 9 1.4 ALTERNATIVES AND IMPROVEMENTS ........................................................ 12 1.5 CONCLUSION ................................................................................................. 15 1.6 FIGURES ......................................................................................................... 17 1.7 REFERENCES ................................................................................................. 20 CHAPTER 2 : A LC-MS PLATFORM FOR IDENTIFYING PROTEIN TARGETS OF SMALL-MOLECULE BINDING RELEVANT TO DISEASE AND METABOLISM .......... 25 2.1 INTRODUCTION .............................................................................................. 25 2.2 DARTS-MS PROTOCOL ................................................................................. 27 2.3 CONCLUSION ................................................................................................. 44 2.4 FIGURES ......................................................................................................... 45 2.5 REFERENCES ................................................................................................. 50 CHAPTER 3 : WHOLE CELL METABOLITE STUDY WITH DARTS-MS ...................... 52 3.1 INTRODUCTION .............................................................................................. 52 3.2 α-KETOGLUTARIC ACID ................................................................................ 55 3.3 α-KETOBUTYRIC ACID ................................................................................... 59 3.4 α-KETOISOCAPROIC ACID ............................................................................ 61 3.5 FIGURES ......................................................................................................... 62 3.6 TABLES ........................................................................................................... 72 3.7 REFERENCES ................................................................................................. 97 CHAPTER 4 : MITOCHONDRIA STUDY WITH DARTS-MS ...................................... 102 vi 4.1 INTRODUCTION ............................................................................................ 102 4.2 α-KETOBUTYRIC ACID ................................................................................. 104 4.3 METFORMIN ................................................................................................. 107 4.4 ROTENONE ................................................................................................... 109 4.5 FIGURES ....................................................................................................... 111 4.6 TABLES ......................................................................................................... 116 4.7 REFERENCES ............................................................................................... 119 CHAPTER 5 : CONCLUSIONS AND FUTURE DIRECTIONS FOR DARTS-MS ........ 120 5.1 CONCLUSION ............................................................................................... 120 5.2 QUANTIFICATION ......................................................................................... 123 5.3 OPTIMIZATION OF SAMPLE PREPARATION .............................................. 126 5.4 FIGURES ....................................................................................................... 129 5.5 REFERENCES ............................................................................................... 130 APPENDIX 1 : STRUCTURE OF TETRAHYMENA TELOMERASE REVEALS PREVIOUSLY UNKNOWN SUBUNITS, FUNCTIONS, AND INTERACTIONS .......... 131 APPENDIX 2 : CHARACTERIZED POST-TRANSLATIONAL PROTEOLYSIS THROUGH DEVELOPMENT OF NEW N-TERMINAL LABELING TECHNIQUES ..... 144 APPENDIX 3 : IMPROVEMENTS IN LC-ESI-MS OF LARGE INTACT PROTEINS BY ADDITION OF VARIOUS SUPERCHARGING AGENTS AS MOBILE PHASE ADDITIVES ................................................................................................................. 150 vii LIST OF FIGURES FIGURE 1-1: SCHEME FOR DARTS ............................................................................ 17 FIGURE 1-2: DARTS PROOF-OF-PRINCIPLE USING FKBP12. ................................. 18 FIGURE 1-3: DARTS USING WHOLE-CELL LYSATE. ................................................ 19 FIGURE 2-1: ENHANCED DARTS-MS PLATFORM. ................................................... 45 FIGURE 2-2: PROTOMAP ANALYSIS. ......................................................................... 46 FIGURE 2-3: TARGET IDENTIFICATION USING GEL-FREE DARTS-MS. ................. 47 FIGURE 2-4: DARTS TARGET IDENTIFICATION BY GELFREE FRACTIONATION VS. WHOLE-SAMPLE SHOTGUN MS. .......................................................................
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