Mass Spectrometry-Based Strategies for Biomolecular Structure Analysis: Steroid Metabolites Structures and Protein-Protein Interactions

Mass spectrometry is an important method for studying the structure of both small molecules and large biomolecules (e.g., proteins). The majority of the applications prior to 1970 were focused on small molecules, owing to the limited ionization methods which posed difficulties in producing gas-phase ions for large biomolecules then. Beginning in the 1980\u27s, with the introduction of new ionization methods (ESI and MALDI), the applications have gradually switched to biological science measuring large bioorganic molecules. Today, with the developing interest in metabolomics and proteomics, and ongoing improvement in MS-based techniques, mass spectrometry is extensively applied in the study of both small and large molecules. The research presented in this thesis falls into two main parts, which focus on the application of MS in (1) structural analysis of steroid metabolites and (2) characterization of protein-protein interactions. In the first part, combinations of different MS methods are adopted and used to solve the structures of unknown steroid metabolites, which are the pheromones responsible for mouse communication in mouse urine. This part includes three chapters, the first two of which discuss the method development of using MS to study the structure of steroid metabolites; and the third chapter presents the application of the MS methods in solving a newly discovered steroid pheromone, which is determined as a sex-specific hormone. In the second part, two MS-based strategies, namely, hydrogen-deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP), are applied in two studies of protein-protein interactions, including: (1) dimerization of SecA, which is a motor protein in bacteria translocation pathway; and (2) interface mapping of EGFR binding to Adnectin1. In the first chapter in Part 2, we used HDX MS to characterize the dimer interface of SecA, and, meanwhile, detected a conformational change from open to closed forms at the pre-protein binding domain upon dimerization. This conformational change provided leads for the active form of SecA. In the second chapter in Part 2, we applied FPOP, which is modified to suit therapeutic protein formulation conditions, to map the epitope of Adnectin1-EGFR interaction at amino acid residue level. The epitope identified agrees with that from both HDX study and crystallography results, presenting more evidence of the capability of FPOP in epitope mapping. These five studies on characterization of steroid metabolites and protein-protein interactions show the successful application of mass spectrometry in the structural study of both small molecules and large proteins. Furthermore, there\u27s a great potential for study of more complex systems