Date of Award
Doctor of Philosophy (PhD)
Mass spectrometry (MS) is an essential tool to study proteins whose structures are of great importance in biological systems. The primary structures of proteins can be determined by the powerful sequencing capabilities of MS. The recent advancements in instrumentation and methodology have made MS increasingly valuable in probing secondary, tertiary and quaternary structures, as well as binding strength, interfaces and in solution dynamics of proteins and protein complexes. Various protein footprinting techniques, including hydrogen-deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP), encode structural information onto the protein molecule in different forms of modifications, and then MS is utilized to interpret the mass shifts resulted from modifications and extract the structural information. Protein footprinting coupled with bottom-up proteomics, which utilizes front-end LC separation and tandem mass spectrometry, has gained a solid ground in protein biophysics. On the other hand, opportunities emerge as native MS, ion-mobility separation, gas-phase activation and fragmentation techniques allow new approaches to be developed. In the first part of this dissertation, we describe epitope mapping of three malaria antigens (Plasmodium vivax Duffy binding protein in Chapter 2, Plasmodium vivax and falciparum cell-traversal protein for ookinetes and sporozoites in Chapter 6) and one flavivirus antigen (West Nile virus envelope protein domain III (DIII) in Chapter 4) by HDX in combination with bottom-up MS. We also report epitope mapping of DIII antigen by FPOP (Chapter 5). Challenged by highly disulfide-linked antigens, sample complexity and discontinuous epitopes with only a few residues each, we implemented immunoprecipitation, non-canonical quenching and digestion protocols to achieve complete sequence coverage and map the epitopes with high confidence and spatial resolution. In the second part (Chapter 3), we describe the usage of native MS and ion mobility to characterize antigen-antibody complexes formed by the Duffy binding protein antigen with various antibodies targeting different epitopes. The last part (chapter 7 and 8) describes the development an on-line HDX, native-spray platform in conjunction with top-down MS. The strategy is validated by determining the amide hydrogen exchange rates of a model peptide at the residue level. With evidence for adequate mixing efficiency, high sequence coverage, low hydrogen scrambling and capable data analysis, we applied the platform to study solution-phase amyloid beta 1-40 monomer structure by continuous-labeling and monitoring exchange kinetics and to probe the dimerization interfaces of human insulin by pulse-labeling experiment. These seven studies demonstrate the applications of the mature bottom-up and promising top-down MS on characterizing protein conformation and protein-protein interactions.
Chair and Committee
Michael L. Gross
Robert E. Blankenship, John-Stephen Taylor, Daved H. Fremont, Hani Zaher
Huang, Yining, "Mass Spectrometry-Based Strategies for Protein Biophysics Higher Order Structure, Interactions and Dynamics" (2016). Arts & Sciences Electronic Theses and Dissertations. 993.
Available for download on Tuesday, December 15, 2116