Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.
Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.
Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Richard W. Vachet
Analytical Chemistry | Biochemistry | Other Biochemistry, Biophysics, and Structural Biology
This dissertation focuses on applying covalent labeling (CL) and mass spectrometry (MS) for characterizing protein-ligand complexes. Understanding protein-ligand interactions has both fundamental and applied significance. Covalent labeling is a protein surface modification technique that selectively modifies solvent-exposed amino acid side chains of proteins. A covalent bond is formed between the functional groups of labeling reagent and protein’s side chain. One of the key factors that affects CL reactivity is a side chain’s solvent accessibility. Ligand binding protects residues on the protein surface from being labeled, and residues involved in ligand binding can be indicated via decreases in labeling extents.
The main goal of this study is to develop strategies that apply CL-MS to characterize protein-ligand complexes. Diethyl pyrocarbonate (DEPC) is the labeling reagent we focused on. First, we developed a strategy that can identify ligand binding site as well as determine the ligand binding affinity to the protein. We characterized the complexes between β-2 microglobulin (β2m) and three amyloid inhibiting molecules under Cu(II)-induced amyloid forming conditions. The rest of the dissertation focused on comparing the information from two complementary MS-based methods, hydrogen deuterium exchange (HDX)-MS and CL-MS. Using three model protein-ligand systems, we demonstrate that the two labeling techniques can provide synergistic structural information about protein-ligand binding when reagents like DEPC are used for CL because of the differences in the intrinsic reaction rates of DEPC-based CL and HDX.
This dissertation highlights the power of CL-MS for characterizing protein-ligand complexes. The understanding of how three amyloid inhibiting molecules bind to Cu(II)-β2M could facilitate future library screening for new drug candidates. Our work also indicates CL-MS is capable of characterizing protein-ligand complexes that are difficult to study by other methods such as X-ray crystallography and nuclear magnetic resonance spectroscopy.
LIU, TIANYING, "Covalent Labeling-Mass Spectrometry for Characterizing Protein-Ligand Complexes" (2020). Doctoral Dissertations. 1847.