University of Kentucky UKnowledge Theses and Dissertations--Toxicology and Cancer Biology Toxicology and Cancer Biology 2018 AN OPTIMIZED SOLID-PHASE REDUCTION AND CAPTURE STRATEGY FOR THE STUDY OF REVERSIBLY-OXIDIZED CYSTEINES AND ITS APPLICATION TO METAL TOXICITY John Andrew Hitron University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/etd.2018.356 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Hitron, John Andrew, "AN OPTIMIZED SOLID-PHASE REDUCTION AND CAPTURE STRATEGY FOR THE STUDY OF REVERSIBLY-OXIDIZED CYSTEINES AND ITS APPLICATION TO METAL TOXICITY" (2018). Theses and Dissertations--Toxicology and Cancer Biology. 22. https://uknowledge.uky.edu/toxicology_etds/22 This Doctoral Dissertation is brought to you for free and open access by the Toxicology and Cancer Biology at UKnowledge. 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John Andrew Hitron, Student Dr. Xianglin Shi, Major Professor Dr. Isabel Mellon, Director of Graduate Studies AN OPTIMIZED SOLID-PHASE REDUCTION AND CAPTURE STRATEGY FOR THE STUDY OF REVERSIBLY-OXIDIZED CYSTEINES AND ITS APPLICATION TO METAL TOXICITY ____________________________________________ DISSERTATION _____________________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By John Andrew Hitron Lexington, Kentucky Director: Dr. Xianglin Shi, Professor of Toxicology and Cancer Biology Lexington, Kentucky 2018 Copyright © John Andrew Hitron 2018 ABSTRACT OF DISSERTATION AN OPTIMIZED SOLID-PHASE REDUCTION AND CAPTURE STRATEGY FOR THE STUDY OF REVERSIBLY-OXIDIZED CYSTEINES AND ITS APPLICATION TO METAL TOXICITY The reversible oxidation of cysteine by reactive oxygen species (ROS) is both a mechanism for cellular protein signaling as well as a cause of cellular injury and death through the generation of oxidative stress. The study of cysteine oxidation is complicated by the methodology currently available to isolate and enrich oxidized-cysteine containing proteins. We sought to simplify this process by reducing the time needed to process samples and reducing sample loss and contamination risk. We accomplished this by eliminating precipitation steps needed for the protocol by (a) introducing an in-solution NEM-quenching step prior to reduction and (b) replacing soluble dithiothreitol reductant with a series of newly-developed high-capacity polyacrylamide-based solid-phase reductants that could be easily separated from the lysate through centrifugation. These modifications, collectively called resin-assisted reduction and capture (RARC), reduced the time needed to perform the RAC method from 2-3 days to 4-5 hours, while the overall quality and quantity of previously-oxidized cysteines captured was increased. In order to demonstrate the RARC method’s utility in studying complex cellular oxidants, the optimized methodology was used to study cysteine oxidation caused by the redox-active metals arsenic, cadmium, and chromium. As(III), Cr(VI), and Cd(II) were all found to increase cysteine oxidation significantly, with As(III) and Cd(II) inducing more oxidation than Cr(VI) following a 24-hour exposure to cytotoxic concentrations. Label-free proteomic analysis and western blotting of RARC-isolated oxidized proteins found a high degree of commonality between the proteins oxidized by these metals, with cytoskeletal, translational, stress response, and metabolic proteins all being oxidized. Several previously-unreported redox-active cysteines were also identified. These results indicate that cysteine oxidation by As(III), Cr(VI), and Cd(II) may play a significant role in these metals’ cytotoxicity and demonstrates the utility of the RARC method as a strategy for studying reversible cysteine oxidation by oxidants in oxidative signaling and disease. The RARC method is a simplification and improvement upon the current state of the art which decreases the barrier of entry to studying cysteine oxidation, allowing more researchers to study this modification. We predict that the RARC methodology will be critical in expanding our understanding of reactive cysteines in cellular function and disease. KEYWORDS: Resin-Assisted Capture, Reversible Cysteine Oxidation, Immobilized Reductants, Cysteine Redox Proteomics, Heavy Metals _____________John Andrew Hitron___________ ________________8-24-2018________________ Date AN OPTIMIZED SOLID-PHASE REDUCTION AND CAPTURE STRATEGY FOR THE STUDY OF REVERSIBLY-OXIDIZED CYSTEINES AND ITS APPLICATION TO METAL TOXICITY By John Andrew Hitron _________Xianglin Shi__________ Director of Dissertation _________Isabel Mellon_________ Director of Graduate Studies ___________8-24-2018__________ Date To my Mom, Dad, and Emily. Thank you for believing in me. ACKNOWLEDGEMENTS This dissertation is an individual work. However no research is conducted in a vacuum, and a multitude of people have supported, facilitated, and mentored me throughout my time as a graduate student. My Doctoral Advisor, Dr. Xianglin Shi, defines the term mentor. Without his support, encouragement, guidance, and most of all patience I could not have completed this work. His dedication to research and, his unshakeable belief in the ideals of science have truly inspired me. I would also like to thank my Dissertation Committee members, Dr. Jia Luo, Dr. Daret St. Clair, and Dr. Hsin-Sheng Yang, as well as my outside reader, Dr. Hollie Swanson, for their insight, support, and encouragement for me. A laboratory is a collaborative environment, and I owe many thanks to my past and present laboratory colleagues, especially Dr. Zhou Zhang, Dr. Xin Wang, Dr. Young- Ok Son, Dr. Lei Wang, Dr. Poyil Pratheeshkumar, and Dr. Senping Cheng for guiding and mentoring me from my first days in the lab until I completed my degree. I would also like to thank Dr. Roy Ram, Dr. Angela Verma, Dr. Olive Ngalame, Yuting Cheng, Kortney Schumann, James Wise, and our ever-patient lab manager Hong Lin. They provided me with different perspectives and new insights into my research, as well as much-needed encouragement on those long days and nights spent in lab. When I did leave the lab, I could always count on my fellow students in the Department of Toxicology and Cancer Biology to help me unwind. I am especially grateful to Drs. Donna Coy, Nikhil Hebbar, and Nathaniel Holcomb. Whether it was letting me bounce ideas of you or just blowing off steam, you all supported me when I needed it most. Without the love and support of my family I would not be the person I am today. I am forever grateful to my mom and dad, Dawn and John Hitron, for instilling me with a strong work ethic and a sense of wonder at the world around us, as well as their unwavering encouragement for me in pursuits. I would also like to thank my sisters Anna and Maggie, as well as their spouses Thomas and John. No acknowledgements would be complete without thanking my beautiful girlfriend Emily Matuszak for her dedication and support over these long years. Words cannot describe how much I appreciate each of these people and their unique places within my life. Finally I would like to thank the institutions that provided funding for my graduate research, including the University of Kentucky and the National Institutes of Environmental Health Sciences. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS………………...……………………………………...…......iii LIST OF TABLES .............................................................................................................. v LIST OF FIGURES ........................................................................................................... vi CHAPTER ONE: INTRODUCTION ................................................................................. 1 CHAPTER TWO: