“Zoom in” on protein functions through integrated mass spectrometry
AdvisorKane, Maureen A.
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AbstractMass spectrometry (MS) has been a powerful tool in cracking the protein codes for human biology regarding to their structure and function. Rich information can be collected through MS either at protein level (native/intact, top-down) or peptide level (middle-down and bottom-up), however integration of these approaches in order to generate a comprehensive view of the protein(s) has been underutilized. In this thesis, strategic integration of MS platforms was developed for two protein systems with the aim of elucidating the fundamental molecular function related to protein-ligand or protein-protein interactions toward fulfilling the potential of MS-based platforms for application in drug discovery. In the first study, the integration of native top down mass spectrometry coupled with ion mobility analysis provided extensive structural information to understand gold finger protein complex that formed with the exchange of zinc. Native top down analysis identified the stoichiometry, binding residues and preferable binding sites when gold replaces zinc in the parent nonclassical zinc finger protein tristetraprolin (TTP). The subtle difference in conformation were monitored by ion mobility simultaneously. The heterogeneity of gold fingers that were reflected by MS-based assays was not obvious by other conventional assays, suggesting the unique analytical power of MS for in-depth drug-target investigations. In the second study, bottom-up and native MS were applied on interrogation of the role of cellular retinol binding protein, type I (CrbpI) in maintaining retinoid homeostasis. The endogenous level of CrbpI was confidently quantified through customized bottom-up assays, which demonstrated a positive correlation with active metabolite retinoic acid (RA). Further investigation focused on the interaction between CrbpI and its biological ligands (retinol and retinal), and the RA-producing enzyme retinal dehydrogenase 1(Raldh1), using native mass spectrometry. These studies contributed fundamental information toward elucidating the role of CrbpI in facilitating RA biosynthesis, and also suggested its potential as a disease marker.
University of Maryland, Baltimore