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Charge Transfer Dissociation Mass Spectrometry of Biomolecules

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Charge Transfer Dissociation Mass Spectrometry of Biomolecules Graduate Theses, Dissertations, and Problem Reports 2017 Charge Transfer Dissociation Mass Spectrometry of Biomolecules Pengfei Li Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Li, Pengfei, "Charge Transfer Dissociation Mass Spectrometry of Biomolecules" (2017). Graduate Theses, Dissertations, and Problem Reports. 6079. https://researchrepository.wvu.edu/etd/6079 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Charge Transfer Dissociation Mass Spectrometry of Biomolecules Pengfei Li Dissertation submitted to the Eberly College of Arts and Sciences at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Glen P. Jackson, Ph. D., Chair Stephen J. Valentine, Ph. D. Suzanne C. Bell, Ph.D. Patrick S. Callery, Ph.D. Peng Li, Ph. D. Department of Chemistry Morgantown, West Virginia 2017 Keywords: Charge Transfer Dissociation, CTD, Ion Activation Technique, Peptides, Insulin, Lipids, Oligosaccharides, HDX Copyright 2017 Pengfei Li ABSTRACT Charge Transfer Dissociation Mass Spectrometry of Biomolecules Pengfei Li Recent advances in many biological disciplines are closely related to the development and application of new mass spectrometry techniques. The investigation of gas-phase ion activation techniques is one of the active research fields. Although researchers have developed a variety of ion activation techniques, they all suffer from certain intrinsic limitations—either limited in the types of fragment ions, or limited by the inefficiency with low charge-state precursor ions. Most of the ion activation techniques are not commercially available, and are at their developing stages. As an attempt to explore new possibilities of fragmenting a gas-phase ion, charge transfer dissociation (CTD) was developed by the Jackson group. CTD is not only workable with low charge state precursor ions (1+ and 2+), but also is workable with highly charged precursor ions (4+, 5+, and 6+). For peptide analysis, CTD produces extensive backbone fragment ions, including a/x, b/y, and c/z ions. Additionally, CTD generates characteristic amino acid side-chain losses, which can complement the sequence information from backbone fragments. An interesting phenomenon of CTD reaction is that the type of predominant fragment ions shifts from a/x to c/z as the precursor charge state increases from 1+ to 3+, or more. For intact insulin analysis, CTD enables the oxidation through a one-electron (dominant) or two-electron (minor) oxidation pathway, which increased the charge state of the intact protein by 1 or 2, respectively. Direct CTD produces a few fragment ions outside the loop defined by disulfide linkages together with charge-increased/charge- decreased species. The MS3-level CID fragmentation of the charge-increased species shows the capability of breaking disulfide linkages, thus provides enhanced structural information. Making use of the ability of being workable with 1+ precursor ions, CTD was employed to fragment phospholipids with various degree of unsaturation. CTD extensively fragments the C-C single bonds within lipid acyl chain, and provides information regarding C=C double bond location. For lipids with various head groups, CTD shows the capability of fragmenting the acyl chains to some extent, but the efficiencies are not suitable for on-line HPLC experiments at this time. CTD was also applied to structural characterization of a methylated linear oligosaccharide, generating both extensive between-ring and cross-ring cleavages. Given the similarity in radical nature between CTD and ETD, CTD was integrated into a HDX workflow to probe the gas-phase conformation of ubiquitin. CTD shows comparable performance to ETD, which demonstrated the potential of CTD in pinpointing the secondary structural elements of gas-phase proteins/peptides. In addition to the exploration to CTD technique, some efforts were devoted to examine the fragmentation behavior of radical cations generated from metastable atom- activated dissociation (MAD) reactions. ESI-generated protonated, sodiated and potassiated POPC ions were firstly subjected to MAD reactions, and then the resulting [POPC]+ ions were further mass-selected and subjected to MS3-level CID reactions respectively. The resulting mass spectra are almost identical—independent of the first generation adducting species. Moreover, the MS3 CID experiment produced extensive fragmentation along the lipid acyl chain, providing valuable structural information associated with C=C double bond positioning. DEDICATION Dedicated to my parents, whose constant love and support made this work possible. iv ACKNOWLEDGEMENTS First of all, I would like to thank my advisor, Dr. Glen Jackson, for devoting his time and efforts to help me on the “journey” to my doctoral degree. He spent a great amount of time in my course discussions, research proposal discussions, analytical seminar rehearsals, research paper preparation and doctoral dissertation preparation. I benefited a lot from his wide range of knowledge—from basic concepts in analytical chemistry, to the hands-on experience of various instruments, to the deep insights into dissociation mechanisms from the physical chemistry perspective. I cannot remember how many times I got inspired from his vivid drawing of instrument schematics or electron-push mechanisms on blackboards, which makes difficult theoretical topics so easy and straightforward and helps me build my own knowledge base. I not only learned the knowledge itself, but also learned the art of mastering knowledge—how to break down difficult concepts and integrate pieces of knowledge into a compact network. Next, I would like to thank Dr. Robert (Bobby) Deimler and Dr. William (Billy) Hoffmann. They were the senior graduate student and postdoc at the time when I joined the group. They spent a great amount time in passing on their experience to me, from the basic steps of operating instruments, to the working principles of a single instrumental unit, as well as important concepts in analytical discipline. They acted like “big brothers” to me: every time I encountered issues in my experiments, they are the first ones I would go to for help. They showed great patience and willingness to coach me. Even if it was a simple calculation formula, they did not mind helping me check each step to fix the error. I would like to thank my other lab mates: Dr. Yan An, Dr. Feng Jin, Kateryna Konstantynova, BohuiLv, Mayara P. V. De Matos, Taylor Krivenki, Ashley Cochran, v Tyler Davidson, Korina Menking-Hoggatt, Zachary Sasiene, Halle Edwards, and Mario Balapuwaduge Mendis, as well as “short-term” members in our laboratory—Heather Birks, Clayton Johnson, Tyler Williams, Iris Kreft and Dr. David Ropartz. All the group members have made contributions in creating a positive and collaborative laboratory atmosphere, in which the lab members help each other in both research work and daily life. All these leave me with fond and valuable memories for my graduate experience. I also would like to thank the members of my dissertation committee—Dr. Stephen Valentine, Dr. Peng Li, Dr. Suzanne Bell and Dr. Patrick Callery. They spent a lot of time reading through my proposal and dissertation, and offered me valuable suggestions to improve both the written and oral work. I would also like to acknowledge the National Institutes of Health (NIH) for the financial support over the course of this work through funding provided by 1R01GM114494-01. Last but not least, I would also like to thank three collaborators—Dr. Gregory Donohoe, Iris Kreft and Dr. David Ropartz. Dr. Gregory Donohoe came up with the idea of coupling CTD technique with on-line HDX workflow, and then we carried this experiment out together on our Bruker amaZon ETD mass spectrometer. His enthusiasm and expertise in peptide and protein chemistry left a strong impression on me. Iris Kreft made a significant contribution to our insulin manuscript by conducting CTD experiments upon insulin and processing data afterwards. It was quite an enjoyable experience to work with her during her six-month stay in our group. Dr. David Ropartz travelled from France to our laboratory in pursuit of a suitable fragmentation technique for fragmenting oligosaccharides. He taught me a lot of fundamentals in sugar chemistry and made it less complicated for me. He showed an impressive ability in arranging and vi documenting experiments in a well-organized way. His passion and dedication to sugar chemistry was very impressive to me, and I really enjoyed working with him. vii Table of Contents ABSTRACT ....................................................................................................................... ii DEDICATION ................................................................................................................
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