Development and Application of Mass Spectrometry-Based Proteomics For: I. Structural Proteomic Guided Investigation of SARS-CoV-2 Polyproteins and Non-Structural Proteins & II. Extending Chemoproteomic Approaches to Decipher the Regulatory Network of LRH-1 [electronic resource]

다국어 초록 (Multilingual Abstract)

Mass spectrometry (MS) is a central analytical technique used to study proteins and biomolecules. It measures mass-to-charge ratio of ions to identify and quantify molecules in simple and complex mixtures. Technological advancement in instrumentation, sample preparation methodologies, and data analysis workflows continue to push the capabilities of MS to answer more complicated questions and vice versa. The work presented in this dissertation applies and extends MS-based technologies across multiple facets of the proteomics landscape including structural proteomic to elucidate the structure of SARSCoV-2 related proteins in Project 1 and chemoproteomic to develop novel methodologies to extend our understanding of the regulatory mechanisms of the nuclear receptor (NR) liver receptor homolog-1 (LRH-1) in Project 2.Structural proteomics uses MS-based methodologies to characterize protein structure. Specifically, it captures the structural plasticity inherent to proteins in solution. In Project 1 we utilized hydrogen deuterium exchange MS and crosslinking MS as complementary techniques to interrogate the published heterotetrameric crystal structures of the SARSCoV-2 nsp7:nsp8 complex. This work revealed that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV nsp7:nsp8 crystal structure, rather they dissociate into a stable dimeric unit that binds to nsp12 in the replication transcription complex without significantly altering nsp7-nsp8 interactions. Subsequently, we generated the first three-dimensional models of the nsp7-10/11 polyproteins and revealed that the nsp7-10/11 structure in complex with Mpro strongly resembles the unbound polyprotein, such that both polyprotein conformation and junction accessibility determine the preference and order of Mpro-mediated cleavages.Chemoproteomic techniques monitor proteome-wide small molecule-protein interactions. Despite advancements in the chemoproteomic field, proteomic analysis of NRs remains a challenge as NRs are relatively low abundant and can be tightly bound to chromatin. In Project 2, our work generated novel probes to capture endogenous LRH-1 and LRH-1 transcriptional complexes to extend our understanding of LRH-1 regulatory mechanisms. Additionally, we made efforts towards the identification of the molecular target of SR1848, to understand how SR1848 represses LRH-1 activity. This led to the discovery of a novel LRH-1 coregulator, peptidyl-prolyl cis-trans isomerases B (PPIB), that we hypothesize regulates LRH-1 trafficking.