BOSTON UNIVERSITY SCHOOL OF MEDICINE Dissertation GLYCAN SEQUENCING AND ISOASPARTATE CHARACTERIZATION BY ELECTRON ACTIVATED DISSOCIATION TANDEM MASS SPECTROMETRY by XIANG YU B.S., Sichuan University, 2007 Submitted in partial fulfillment of the requirements for the degree of Doctor of Biochemistry 2013 Approved by First Reader Catherine E. Costello, Ph.D. Professor of Biochemistry Second Reader Cheng Lin, Ph.D. Assistant Professor of Biochemistry ii © Copyright by XIANG YU 2013 iii ACKNOWLEDGEMENTS First of all, I would like to express my deepest gratitude to my advisor, Professor Catherine E. Costello. I would like to thank you for your guidance and support throughout my Ph.D. study. You have always been inspiring and thoughtful in science, patient and progressive as a mentor. You are one of the best advisors I met, very knowledgeable, resourceful, approachable, and extremely nice to your students. You have been truly a role-model for me, in both scientific research and personality. I am also grateful to Professor Cheng Lin for working closely with me for the last five years. You have lots of great ideas and together we translated them into many different successful projects. You have been always available to answer my questions inside and outside science. Your enthusiasm in scientific excellence gives me confidence in pursuing a career as a scientist. I want to thank Professor Peter B. O’Connor, who introduced me into the world of mass spectrometry at my second year in graduate school. Although I had worked with you for only one year, you have greatly enlightened my interest in mass spectrometry. I would like to thank Professor Joseph Zaia, Professor David Harris, and Professor Karen Allen for spending your valuable time serving on my committee and giving me numerous constructive advices. iv Many current and past members in the Center for Biomedical Mass Spectrometry (CBMS) contributed to this thesis and it is a great pleasure to work with all of you. Special gratitude goes to Professor Mark Mccomb and Christian Heckendorf, who provided me a strong IT support; Dr. Yiqun Huang, who helped me a lot on physical chemistry; Dr. Weidong Cui, who taught me how to maintain and trouble-shoot a mass spectrometer; Dr. Chunxiang Yao, with whom I did my first mass spectrometry experiment; Dr. Xiaojuan Li, who worked together with me on the top-down project; Yu Huang, who collaborated with me on the GAG EDD project; Prof. Sandrine Voillard-Bourgoin, who assisted me on FT-ICR mass spectrometry; Dr. Xiaofeng Shi, from whom I learnt a lot on glycan chemistry. I would like to thank the people who helped me at different stages of my doctorate study. They are Ying Zhou, Yang Mao, John Haserick, Han Hu, Liang Han, YanYan Lu, Kshitij Khatri, Mengdi Fan, Chun Shao, Yuhuan Ji, Yi Pu, Dr. Stephen Whelan, Dr. Deborah Francoleon, Dr. Hye Jung Park, Dr. Amanuel Kehasse, Dr. Yan Jiang, Dr. Greg Staples, Dr. Roger Theberge, Dr. Edwin Motari, Dr. Jason Cournoyer, Dr. Zhenning Hong and Dr. David Perlman. I would also thank Denise Neves and Patricia Bullock for all your administrative efforts to let me focus on my research. It is also a great pleasure to work as an intern for Dr. Wendy Zhong in the Merck research laboratory. I learnt a lot from you on things that I never have a chance to learn in school. v Most importantly, my best friends, Dr. Yi Yang, Yu Huang, Dr. Zhenxiang Xi, Xiaobin Xu, and Dr. Songmao Zheng. I’m so blessed to have all of you in my life. Finally, I want to thank my parents, for their utmost love. vi GLYCAN SEQUENCING AND ISOASPARTATE CHARACTERIZATION BY ELECTRON ACTIVATED DISSOCIATION TANDEM MASS SPECTROMETRY (Order No.) XIANG YU Boston University, School of Medicine, 2013 Major Professor: Catherine E. Costello, Ph.D., Professor of Biochemistry ABSTRACT In this study, we carefully examined several types of electron activated dissociation (ExD) processes and developed new ExD techniques that should facilitate biological research, placing particular emphasis on glycan and protein characterization. The first part of this study focuses on determination of ExD fragmentation mechanisms and application of ExD to glycan de novo sequencing. Through variation of the electron energy and metal charge carriers, the behaviors of model glycans were systematically studied and a new ExD fragmentation process, designated as electronic excitation dissociation (EED), was found to be the most informative. By identifying and controlling the key parameters, we improved the EED efficiency, to a level that now allows EED to be performed on a time scale that is compatible with high performance liquid chromatography scale. Theoretical modeling was employed to gain insights into the charge remote fragmentation behavior vii inherent in the EED process. The experimental results demonstrated that EED has the potential to provide the experimental basis for high-throughput, de novo glycan sequencing. The second part of this study focuses on the determination of deamidation of asparagine residues and isomerization of aspartate residues within proteins. In order to avoid the generation of artifacts during trypsin digestion, a comprehensive top-down ExD method was developed to identify both asparagine deamidation and isoaspartate formation at the level of the intact protein With the consideration that the top-down strategy will eventually fail for high molecular weight proteins, a middle-down ExD method was next developed, for the analysis of peptides generated by proteolysis with Staphylococcal serine protease Protease V8 (Glu-C), carried out at slightly acidic conditions. In addition, the potential for use of in-source decay in isoaspartate analyses was evaluated and its fragmentation mechanisms were investigated. This research establishes new tools for structural determinations of glycans and significant improvements in methods for the isomer- and site-specific analysis of proteins that contain Asp or Asn residues that can undergo conversion to isoAsp, and provides insight toward understanding and controlling the fundamental processes that lead to the types of fragment ions observed in electron activated dissociation mass spectra. viii TABLE OF CONTENTS Chapter 1 Introduction..................................................................................................1 1.1 Introduction to mass spectrometry...........................................................................1 1.1.1 Ion sources...................................................................................................1 1.1.2 Mass analyzers.............................................................................................4 1.1.3 Ion activation methods for tandem mass spectrometry (MS/MS).............13 1.2 Glycan sequencing by electron activation dissociation.........................................19 1.2.1 Glycan chemistry and its biological roles..................................................19 1.2.2 Tandem mass spectrometry of glycans......................................................23 1.2.3 Nomenclature of glycan fragments............................................................24 1.2.4 Electron activated dissociation of glycans.................................................27 1.3 Overview of glycan sequencing by electron activated dissociation......................32 1.4 Isoaspartate characterization by electron activated dissociation...........................33 1.4.1 Asparagine deamidation and aspartate isomerization................................33 1.4.2 Mass spectrometry identification of deamidation and isoaspartate formation....................................................................................................34 1.5 Overview of isoAsp identification by electron activated dissociation...................36 1.6 Summary................................................................................................................37 Chapter 2 Energy-Dependent Electron Activated Dissociation of Metal-Adducted Permethylated Oligosaccharides................................................................38 ix 2.1 Introduction............................................................................................................38 2.2 Experimental..........................................................................................................42 2.2.1 Materials....................................................................................................42 2.2.2 Reducing End Reduction...........................................................................42 2.2.3 Reducing End 18O-Isotope Labeling..........................................................43 2.2.4 Permethylation...........................................................................................43 2.2.5 Electrospray Conditions.............................................................................44 2.2.6 Tandem Mass Spectrometry......................................................................44 2.2.7 Data Analysis.............................................................................................45 2.3 Results and Discussion..........................................................................................46 2.3.1 ExD of the Sodium-Adducted Permethylated Maltoheptaose...................46 2.3.2 ExD of Lithium-Adducted Permethylated Maltoheptaose........................57 2.3.3 ExD of Alkaline Earth Metal-Adducted Permethylated Maltoheptaose............................................................................................63