ORCID
http://orcid.org/0000-0002-3050-8038
Date of Award
Spring 5-15-2022
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Mass spectrometry (MS)-based methods are efficacious tools to probe protein higher order structure (HOS). In this work, we describe MS-based footprinting methods that impart covalent modifications to protein backbone and amino acid sidechains, allowing for the differential measurement of mass additions by MS to monitor protein dynamics. Chapter 1 includes an excerpt from a book chapter highlighting the current state of research on the development and application on specific amino acid footprinting MS in structural proteomics using MS. Hydrogen deuterium exchange (HDX)-MS and fast photochemical oxidation of proteins (FPOP)-MS are also introduced. We next perform problem-driven footprinting method development. Residues Ser and Thr are biologically important owing to their high abundance on paratope-epitope interactions and as sites of phosphorylation, but they are challenging to footprint owing to their weak nucleophilicity. Chapter 2 introduces benzoyl transfer for footprinting alcohol-containing residues in higher order structural (HOS) applications of MS-based proteomics. Using cyclopeptides as part of a workflow for validating footprinting reagents, we learned most importantly that benzoyl fluoride (BF) modifies weakly nucleophilic Tyr at a rate 10x greater than the current state-of-the-art nucleophile footprinter, diethyl pyrocarbonate (DEPC). Through an extended evaluation of BF, we outline and propose a systematic workflow for validating specific amino acid footprinting MS reagents for protein HOS elucidation. Specific amino acid footprinting is still performed by hand despite advances in technology that afford robotic automation of other techniques such as HDX-MS. We repurposed a Trajan PAL LEAP hydrogen-deuterium exchange MS (HDX-MS) robot for amino acid specific footprinting MS (Chapter 3-1), demonstrating the method’s efficacy in measuring with high precision changes in protein dynamics upon ligand binding. This method lays the groundwork for performing, for the first time, footprinting reagent discovery and both top down and bottom up proteomics experiments entirely through robotic automation. The Covid19 pandemic introduced challenges in diagnostics for which protein footprinting may support. Motivated by the ability of MS-based methods to rapidly detect and quantify proteins (i.e., antigenic SARS-CoV-2 proteins and host antibodies), we collaborated with Professor Jonathan Barnes group and Dr. David Hambly at Advanced Therapy Consulting, Inc., to develop small molecule reporters for SARS-CoV-2 diagnostics by specific amino acid footprinting MS. We demonstrate that an imidazolium compound modifies IgG1 antibodies detectable by laser desorption/ionization MS (LDI-MS) at levels as low as 0.9 fmol. These data motivate the use of covalently attached reporter molecules containing metals for inductively coupled plasma (ICP)-MS studies, which should have even lower detection limits. The final method development project characterizes temperature induced protein HOS perturbation using HDX-MS and fast photochemical oxidation of proteins MS (FPOP-MS). Biotherapeutic quality control is critical for monitoring treatment efficacy, and forced degradation studies are typically performed on intact proteins, preventing a complete understanding of local changes in protein dynamics. In a collaboration with Dr. Ying Zhang’s group at Pfizer, Inc, we demonstrate that FPOP-MS is more sensitive than HDX-MS in measuring small, local changes in HOS for the biotherapeutic antibody bevacizumab, and we observe several distinct phenomena occurring throughout the protein sequence as a function of forced degradation time. Our findings motivate the adoption of FPOP-MS for quality control analysis by pharmaceutical companies to complement limited HDX-MS. The latter portion of my dissertation is focused on the application of MS-based techniques to elucidate mechanisms of disease. The Covid19 pandemic has caused significant devastation to society, motivating the study of fundamental biophysical characterization of SARS-CoV-2 viral replication. SARS-CoV-2 nucleocapsid (N) protein is a structural protein for which residues involved in RNA binding and phosphorylation can affect viral load. Chapter 5 describes a collaboration with Professors Gaya Amarasinghe and Daisy Leung, (1) mapping the RNA binding site of SARS-CoV-2 nucleocapsid protein using HDX-MS and (2) developing and publishing a protocol for characterizing RNA binding viral proteins by HDX-MS. Here, we validate putative RNA binding residues in the N-terminal domain and discover, for the first time, a putative binding site in the SR-rich linker region of the protein. The experiment was challenged by phase separation decreasing solubility and thereby reducing protein digestion and coverage. The findings motivate method development to study phase separating viral proteins. Finally, the SARS-CoV-2 pandemic underscores the need to elucidate proactively fundamental biophysics and host-pathogen interactions of emerging diseases. Rift Valley Fever Virus (RVFV) is a zoonotic pathogen with pandemic potential. Chapter 6 outlines a collaboration with Professors Gaya Amarasinghe and Daisy Leung, applying a method using size exclusion chromatography (SEC)-native MS for rapid quality control prior to footprinting MS and using it to characterize the newly discovered glycosylated host entry factor (LRP1) for RVFV. LRP1 affords viral entry regardless of glycosylation state, and our data lay the groundwork for footprinting studies of LRP1 with RVFV antigenic proteins. Taken together, this work underscores the imperative for problem-driven method development in protein footprinting. The conclusion in Chapter 7 highlights limitations, challenges, and future perspectives, including a list of nine objectives for the continuation of these studies and the advancement of protein footprinting applied to increasingly critical biological problems.
Language
English (en)
Chair and Committee
Michael L. Gross
Committee Members
Timothy A. Wencewicz
Recommended Citation
Moyle, Austin Blake, "Mass Spectrometry-based Biophysics of Protein Assemblies, Antibodies, and Antigens: Method Development and Applications" (2022). Arts & Sciences Electronic Theses and Dissertations. 2723.
https://openscholarship.wustl.edu/art_sci_etds/2723