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Development of Ion Mobility Mass Spectrometry Instrumentation to Investigate the Gas-Phase Structures of Protein and Protein Complex Ions

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Development of Ion Mobility Mass Spectrometry Instrumentation to Investigate the Gas-Phase Structures of Protein and Protein Complex Ions Development of Ion Mobility Mass Spectrometry Instrumentation to Investigate the Gas-Phase Structures of Protein and Protein Complex Ions Samuel J. Allen A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2017 Reading Committee: Matthew F. Bush, Chair Robert E. Synovec Joshua C. Vaughan Program Authorized to Offer Degree: Chemistry ©Copyright 2017 Samuel J. Allen 1 University of Washington Abstract Development of Ion Mobility Mass Spectrometry Instrumentation to Investigate the Gas-Phase Structures of Protein and Protein Complex Ions Samuel J. Allen Chair of Supervisory Committee: Assistant Professor Matthew F. Bush Department of Chemistry This dissertation reports the development of new ion mobility mass spectrometry (IM- MS) instrumentation to analyze protein and protein complex ions. IM-MS is a gas-phase analytical technique that separate ions based on their collision cross section (a description of ion shape) and mass-to-charge ratio. Electrospray ionization of samples from buffered solutions at biologically relevant pH generates “native-like” protein ions, which retain noncovalent interactions and compact conformations. IM-MS analysis enables the determination of the shape and assembly of native-like ions, which can be used to infer information about the solution conformations of biomolecules. New IM devices were developed to improve the informational content obtained from IM-MS experiments. First, an RF-confining drift cell was developed and 2 implemented in a commercial mass spectrometer. Experimental results and ion trajectories implemented using SIMION were used to describe the separation principles of ions in RF- confining drift cells. Those results show that RF-confining drift cells separate ions similarly to traditional IM devices and that applied RF potentials have minimal effects on the effective temperatures of gas-phase ions. The RF-confining drift cells was used to report collision cross sections for 349 ions, 155 of which are for ions that have not been characterized previously using IM. The effects of ionization conditions and analyte solutions on the charge states and collision cross sections of ions was also investigated. An additional IM device based on Structures for Lossless Ion Manipulations (SLIM) was developed. SLIM is an emerging IM technology that can be implemented as modular platforms to perform ion separations, filtering, and trapping. The first collision cross sections determined using SLIM are reported. IM analysis of native-like protein ions shows that those ions exhibit significant structural heterogeneity in the gas-phase. To evaluate the stability and dynamics of native-like protein ions, IM-IM-MS functionality was implemented into the SLIM device. Dynamic gas-phase conformations were observed for native- like protein ions, which has significant implications for the broader community’s interpretation of IM-MS results. The stability of structural subpopulations of native-like ions was investigated as a function of gas-phase ion lifetime at near-ambient temperatures. The conformations of subpopulations evolve continuously in the gas-phase, but even after 4 seconds, the subpopulations exhibit different distributions of collision cross sections. This suggests that native-like ions in IM-MS experiments can retain some memory of their initial gas-phase structures for up to seconds at near-ambient temperature. 3 Dedication To my family. 4 Acknowledgements I would like to thank my advisor, Prof. Matthew F. Bush, for the extensive mentorship and guidance during my PhD. He gave me the freedom to pursue what I wanted and provided numerous opportunities to advance my career. I would also like to thank Prof. Robert Synovec, Joshua Vaughan, James Bruce, Frantisek Turecek, Dustin Maly, Bo Zhang, and Daniel Chiu for serving on my academic committees and providing valuable discussions related to my research. The instrumentation that I developed was enabled by tremendously experienced and helpful engineers at the University of Washington. Bob Scott (now at UW Physics - Instrument Shop Manager), John Heutnik (retired, UW Chemistry - Instrument Maker), Ed McArthur (retired, UW Chemistry - Instrument Maker), and Pat McCrory (UW APL - Machine Shop Manager) fabricated the majority of the SLIM instrument. Eric Strakbein (UW Chemistry - Facilities and Machine Shop Manager) and Brian Wadey (UW Chemistry - Instrument Maker) were extremely helpful and responsive with design considerations and fabrication of a new instrument that I helped design. Electronics Shop research engineers Lon Buck, William Beaty, and Roy Olund created several electronics that were used in instruments I developed, and were always present to loan test equipment or impart expert electrical engineering advice. I would also like to thank the SLIM consortium at Pacific Northwest National Laboratory, including Dr. Richard Smith, Dr. Yehia Ibrahim, Dr. Randy Norheim, Dr. Tsung-Chi Chen (now at ThermoFisher Scientific), Spencer Prost, and Dr. Ian Webb for sharing designs, software, and technical expertise. I would also like to specifically thank Dr. Gordon Anderson (GAA Custom Engineering) who was present for the first ion signal the SLIM instrument and was always available to assist me in troubleshooting. Kevin Giles and Tony Gilbert (Waters Corporation) fabricated the RF-confining drift cell and helped me obtain my first ion mobility results. 5 I would also like to thank UW Chemistry’s Lochlan Hickok (Stockroom Manager), Michael Zimmerman (Fiscal Specialist), Elizabeth Lee (Fiscal Specialist), and Susan Harmon (previous Purchasing Agent) for being helpful with my many inquiries and for ensuring that I was able to purchase and receive the incredible amount of materials required in my graduate work. Ashley Zigler (previous Graduate Program Coordinator), Diana Knight (Assistant to the Chair), and Krista Holden (Graduate Program Coordinator) were prompt to reply to questions about the program, and were helpful in navigating all the processes involved in obtaining my PhD. I would also like to thank the past and present members of the Bush Lab for the insightful discussions and debates that contributed to my growth as a scientist. They served as colleagues in science and in life, and I will cherish the time I was able to spend with them all. My time in graduate school was enriched by my close friends Dr. Sam Marionni and Ken Laszlo with whom I shared too many memories to recount here. Rae Eaton was instrumental in the development of the SLIM instrument, and we shared the joy and tears associated with building a scientific instrument. I am also grateful for the extremely talented and hard-working students (Alicia Schwartz, Cindy Wei, and Chrissy Stachl) that greatly contributed to my projects. California State University, Chico Prof. Lisa Ott, Erik Wasinger, Randy Miller, Chris Nichols, and Dan Edwards motivated me to apply to graduate school. Their passion for chemistry and teaching was inspiring and I will always appreciate the time they spent helping me “learn how to learn”. This journey would not have been possible without the love and support of my family and friends. From missed birthdays, cancelled trips, and long bouts of silence, they have shown the greatest patience for my dreams. 6 Lastly, thank you to my wife, Sonja. In the chaos that is graduate school, she gave me clarity, compassion, and our beautiful son Leo. This time in graduate school will be a footnote in the grander story of our lives. 7 Nomenclature CRM Charged-Residue Model Da Dalton, the unified atomic mass unit DC Direct Current e elementary charge, 1.6021766208(98) x 10–19 C E Electric Field electrodynamic referring to time-dependent electric potentials electrostatic referring to time-independent electric potentials E∙P–1 drift field strength: electric field divided by pressure, V∙cm–1∙Torr–1 ESI Electrospray Ionization He helium, in reference to IM buffer gas IM Ion Mobility –23 –1 kB Boltzmann’s constant, 1.38064852(79) x 10 J∙K μ reduced mass, often between ion and neutral masses MS Mass Spectrometry m/z mass-to-charge ratio, the standard measurement unit in MS N2 nitrogen, in reference to IMS buffer gas Ω ion-neutral collision cross section RF Radio-Frequency, referring the frequency of AC potentials SIMION Ion and Electron Optics Simulator SLIM Structures for Lossless Ion Manipulations tD drift time Teff Effective Temperature TOF time-of-flight, an MS analyzer VPP Peak-to-Peak Voltage, referring to the amplitudes of RF potentials z ion charge state 8 Table of Contents Abstract ........................................................................................................................................... 1 Dedication ....................................................................................................................................... 3 Acknowledgements ......................................................................................................................... 4 Nomenclature .................................................................................................................................. 7 Table of Contents ............................................................................................................................ 8 1. Introduction ............................................................................................................................... 12 1.1 General Overview ......................................................................................................
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