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High Resolution Mass Spectrometric Approaches to Study Protein Structure and Environment in Lyophilized Solids Lavanya Iyer Purdue University

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High Resolution Mass Spectrometric Approaches to Study Protein Structure and Environment in Lyophilized Solids Lavanya Iyer Purdue University Purdue University Purdue e-Pubs Open Access Dissertations Theses and Dissertations January 2015 High Resolution Mass Spectrometric Approaches To Study Protein Structure and Environment in Lyophilized Solids Lavanya Iyer Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations Recommended Citation Iyer, Lavanya, "High Resolution Mass Spectrometric Approaches To Study Protein Structure and Environment in Lyophilized Solids" (2015). Open Access Dissertations. 1416. https://docs.lib.purdue.edu/open_access_dissertations/1416 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School Form 30 Updated 1/15/2015 PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Lavanya Kannan Iyer Entitled High Resolution Mass Spectrometric Approaches To Study Protein Structure And Environment In Lyophilized Solids For the degree of Doctor of Philosophy Is approved by the final examining committee: Elizabeth M. Topp Chair Gregory T. Knipp Lynne S. Taylor Steven L. Nail To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material. Approved by Major Professor(s): Elizabeth M. Topp Approved by: Elizabeth M. Topp 8/28/2015 Head of the Departmental Graduate Program Date i HIGH-RESOLUTION MASS SPECTROMETRIC APPROACHES TO STUDY PROTEIN STRUCTURE AND ENVIRONMENT IN LYOPHILIZED SOLIDS A Dissertation Submitted to the Faculty of Purdue University by Lavanya K Iyer In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2015 Purdue University West Lafayette, Indiana ii For my parents and grandparents iii ACKNOWLEDGEMENTS I would like to thank my major advisor, Dr. Elizabeth M. Topp. She has been an excellent mentor. I appreciate the freedom and encouragement she gave me to explore different ideas. I will always value the time I spent in her group, learning about great science. I thank my committee members, Dr. Greg Knipp and Dr. Lynne Taylor for their scientific insight and am indebted to Dr. Steve Nail, who gave me the opportunity to intern at Baxter Biosciences. It was a wonderful learning experience. I thank Dr. Andreas Sophocleous, who was a great source of inspiration when I started my graduate work. His scientific expertise, strong work ethic and willingness to share knowledge are qualities that I try to incorporate in my life. Many thanks to Dr. Shenbaga Moorthy Balakrishnan for being a great teacher and for interesting discussions about science and cricket. I am grateful to Dr. Saradha Chandrasekhar, Dr. Jun Zhang, Ehab Moussa and Anshul Mishra for being excellent lab mates. I would also like to thank Mary Ellen Hurt, Nancy Cramer and Janine Mott, for making graduate life smoother. I will forever be grateful to my parents, Geetha and Kannan and my sister Madhuri for their unconditional love and support throughout life. They inspire me to work hard, be honest and be brave enough to try new things. I owe a great deal to my grandparents, V. Ramaswamy and Kalyani Ramaswamy for reminding me to have faith, continue learning and be a good person. Special thanks to my aunt, Lalitha Shankar, for being a pillar of strength. I want to express my gratitude to my wonderful parents-in-law, Minaxi and Piyushkumar Panchal, whose iv encouragement kept me going. Finally, I would like to thank my husband, Jainik Panchal, for his endless patience, love and support. v TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................................... viii LIST OF FIGURES ........................................................................................................................... x LIST OF ABBREVIATIONS ........................................................................................................... xiv ABSTRACT .................................................................................................................................... xv CHAPTER 1. INTRODUCTION.................................................................................................... 1 1.1 PROTEIN AGGREGATION IN THE SOLID STATE ........................................................ 1 1.2 COVALENT LABELING OF PROTEINS .......................................................................... 3 1.2.1 Chemical Labeling Agents ..................................................................................... 3 1.2.2 Photolytic Labeling Agents ..................................................................................... 4 1.2.2.1 Arylazides ............................................................................................................... 4 1.2.2.2 Diazirines ............................................................................................................... 5 1.2.2.3 Benzophenones ..................................................................................................... 6 1.3 CROSSLINKING OF PROTEINS ..................................................................................... 7 1.4 ADVANTAGES OF MODIFICATION USING PAAs ....................................................... 10 1.5 ADVANTAGES OF CROSSLINKING ............................................................................. 10 1.6 SPECIFIC AIMS ............................................................................................................. 11 1.7 OVERALL APPROACH .................................................................................................. 13 1.8 REFERENCES ............................................................................................................... 15 CHAPTER 2. PHOTOLYTIC LABELING TO PROBE MOLECULAR INTERACTIONS IN LYOPHILIZED POWDERS .................................................................................. 17 2.1 ABSTRACT .................................................................................................................... 17 2.2 INTRODUCTION ............................................................................................................ 18 2.3 MATERIALS AND METHODS ....................................................................................... 20 2.3.1 Sample Preparation ............................................................................................. 21 2.3.2 Photolytic Labeling and MS Analysis of Intact Protein......................................... 22 2.3.3 Effect of Irradiation Time and pLeu concentration on Labeling Efficiency ........... 23 2.3.4 MS- and MS/MS- Analysis of Labeled apoMb Peptides ...................................... 24 2.3.5 Formulation Effects .............................................................................................. 24 vi Page 2.3.6 Photolytic Labeling with p-Benzoyl-L-Phenylalanine (pBpA) ............................... 25 2.3.7 MS/MS Analysis of GCG (1-8)* Dimer ................................................................. 25 2.4 RESULTS ....................................................................................................................... 26 2.4.1 Intact Protein Labeling ......................................................................................... 26 2.4.2 Labeling Kinetics .................................................................................................. 27 2.4.3 Peptide Labeling .................................................................................................. 28 2.4.4 MS/MS Analysis of Peptide L32-K42 (LFTGHPETLEK) ...................................... 32 2.4.5 Formulation Effects .............................................................................................. 33 2.4.6 Photolytic Labeling with p-benzoyl-L-phenylalanine (pBpA) ................................ 34 2.4.7 MS/MS Analysis of GCG (1-8)* Monomer and Dimer.......................................... 36 2.5 DISCUSSION ................................................................................................................. 37 2.6 CONCLUSIONS ............................................................................................................. 42 2.7 REFERENCES ............................................................................................................... 43 CHAPTER 3. PHOTOLYTIC CROSSLINKING TO PROBE MOLECULAR INTERACTIONS IN LYOPHILIZED SOLIDS ....................................................................................... 46 3.1 ABSTRACT .................................................................................................................... 46 3.2 INTRODUCTION ............................................................................................................ 47 3.3 MATERIALS AND METHODS ....................................................................................... 51 3.3.1 Sample Preparation ............................................................................................. 52 3.3.2 Labeling Mb with SDA in Solution ........................................................................ 52 3.3.3 Structural Integrity of Labeled Protein ................................................................
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