ORCID
https://orcid.org/0000-0002-6365-4672
Date of Award
12-17-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Proteins are critical for most biological functions, and the three-dimensional structure of a protein, called higher order structure (HOS), determines its function. Elucidating the HOS and dynamics of a protein has been a major area of research in structural biology and proteomics, as the structural determination provides insight into the protein’s biological role. Mass spectrometry (MS)-based methods have become increasingly utilized to investigate protein HOS and interactions with various ligands, and protein footprinting MS is one example of structural proteomics that has been extensively employed. Protein footprinting covalently attaches a mass tag (or modification) onto the protein, and then, by MS identification, locates the sites of the protein that underwent modification. By determining the modified residues, MS-based footprinting reveals the solvent accessible surface area (SASA) of the protein, reflecting protein HOS. There are several different reagents and strategies to footprint proteins that elucidate the HOS. In Chapter 1, many footprinting MS strategies are discussed in detail, including Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS), Radical-Based Footprinting, Specific Amino Acid Footprinting, and Cross-Linking Mass Spectrometry (XL-MS), as well as other MS-based structural proteomics methods, such as Native Mass Spectrometry (nMS) and Ion Mobility Mass Spectrometry (IM-MS). The second section of the dissertation emphasizes the development and determination of appropriate conditions for specific amino acid footprinting reagents. Chapter 2 establishes a protocol to a validate new footprinting reagents by identifying reactive residues, determining an appropriate quench, and examining reagent specific considerations (e.g., pH). The validation workflow was implemented for a new footprinting reagent, benzoyl fluoride (BF), and an established reagent, diethyl pyrocarbonate (DEPC). Chapter 3 investigates the efficacy of various methods, both MS-based and spectroscopic methods, in evaluating footprinting-induced perturbation of protein HOS. Specific amino acid footprinting can produce HOS changes to the protein under certain conditions (e.g., high footprinter concentrations), causing modification on non-native structures; therefore, establishing appropriate footprinting conditions is necessary to ensure that the structure of the protein remains native during footprinting. The third section focuses on aggregation amyloid beta (Aβ), which is one of the major features of Alzheimer’s Disease (AD), as well as investigating various ligand binding partners with Aβ. Chapter 4 reviews the principles of protein aggregation and applications of various MS-based methods (i.e., nMS, IM-MS, Continuous HDX, Pulsed HDX, Fast Photochemical Oxidation of Proteins (FPOP) and other irreversible labeling methods, and XL-MS) in studying Aβ aggregation and determining inhibitors of Aβ aggregation. Chapter 5 utilizes DEPC footprinting to evaluate Aβ aggregation at the intact protein level and residue level, specifically highlighting the N-terminal region of Aβ during aggregation. The interaction between oligomeric Aβ and soluble triggering receptor expressed on myeloid cells 2 (sTREM2) is investigated in Chapter 6 by using DEPC footprinting and XL-MS, finding that sTREM2 disrupts the HOS of oligomeric Aβ and that binding occurs at the N-terminal region of Aβ. The last section of the dissertation centers on the HOS and dynamics of a membrane protein, Vitamin K Epoxide Reductase (VKOR), binding to a small molecule ligand, warfarin. Chapter 7 employs HDX-MS to assess the binding interaction between VKOR in a nanodisc construct and warfarin. HDX-MS identifies regions VKOR that undergoes a significant decrease in deuterium uptake in the presence of warfarin, indicating protection and less dynamics of VKOR when bound to warfarin. These seven chapters display the importance of MS-based footprinting in structural proteomics. MS-based footprinting can provide vital information on protein HOS and ligand binding interactions that may be extremely difficult to characterize with any other biochemistry-based method. MS-based strategies are positioned to play a pivotal role in structural proteomics moving forward, and developing new footprinting approaches is critical for further application of these methods.
Language
English (en)
Chair and Committee
Michael Gross
Committee Members
Meredith Jackrel; Michael Gross; Thomas Brett; Timothy Wencewicz; Weikai Li
Recommended Citation
Wagner, Wesley, "Validation and Application of Mass Spectrometry-based Methods for Probing Protein Higher Order Structure" (2024). Arts & Sciences Electronic Theses and Dissertations. 3372.
https://openscholarship.wustl.edu/art_sci_etds/3372