Ultraviolet Photodissociation Mass Spectrometry for Characterization of Biological Molecules

摘要。了解脂质，蛋白质，甚至更大的大分子组件的功能

Abstract. Understanding the functions of lipids, proteins and even larger macromolecular assemblies depends on deciphering complex structures of individual molecules as well as decrypting how those molecules interact, often via networks of non-covalent interactions. In order to advance the elucidation of biomolecular organization and functional outcomes, new methods are needed to push the limits of structural insight, providing more detailed holistic chemical information with greater sensitivity. The critical interplay between structure/function is evidenced in numerous biologically-motivated problems, ranging from understanding the ways that pathogenic bacteria develop antibiotic resistance to the design of new drugs that selectively bind and inhibit the functions of protein targets. The ongoing need for even greater chemical insight has motivated my group’s effort to develop innovative mass spectrometry methods to characterize structures of biological molecules in unprecedented detail, especially lipids and proteins which are featured in this proposal. The overarching goal of my research program is to develop state-of- the-art tandem mass spectrometry technologies, particularly highlighting ultraviolet photodissociation (UVPD) and hybrid MS/MS methods, for structural elucidation of lipids, proteins, and protein complexes. These new methods will be showcased for solving challenging problems in three areas. (1) Lipids: (i) profiling lipids of pathogenic bacteria and their signatures of antibiotic resistance, and (ii) structural characterization of unsaturations, oxidations and modifications of lipids that occur during remodeling of cellular membranes. (2) Protein complexes: (i) characterization of protein-ligand complexes, membrane protein complexes, protein/nucleic acid complexes, and macromolecular assemblies, and (ii) advancing capillary electrophoresis for native separations and exploration of the interactome. (3) Post-translational modifications: focusing on decoding the phosphorylation patterns of the C-terminal domain of RNA polymerase II which regulates transcription. These high impact problems are supported via numerous collaborations with microbiology and molecular biology groups who recognize the value of frontier mass spectrometry strategies for elevating biomedical research. This supplement supports acquisition of an ExD cell to enable electron-based activation on existing mass spectrometers and a solvent evaporator to accelerate sample preparation workflows. The ExD cell will support research areas 2 and 3 by allowing electron capture dissociation and charge reduction capabilities for analysis of post-translational modifications and cleavage of disulfide bonds in proteins. The solvent evaporator will facilitate preparation of samples in all three research areas.