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

https://orcid.org/0000-0002-0450-5570

AccessType

Campus-Only Access for One (1) Year

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2021

Month Degree Awarded

September

First Advisor

Richard Vachet

Second Advisor

Stephen Eyles

Third Advisor

Ricardo Metz

Fourth Advisor

Igor Kaltashov

Subject Categories

Biochemistry, Biophysics, and Structural Biology

Abstract

This dissertation focuses on the use of mass spectrometry (MS) to study therapeutic protein higher order structure (HOS) by encoding the structure into the mass of the protein. As therapeutic proteins become more common in the pharmaceutical industry, the need for methods that accurately determine their HOS has grown. Two methods applied here are hydrogen deuterium exchange (HDX) MS and diethylpyrocarbonate (DEPC) covalent labeling (CL) MS. We demonstrate how these two methods provide complementary, and sometimes synergistic, information about protein HOS. HDX/MS reports on both changes in solvent exposure and changes in protein dynamics, and as a result it can often lead to ambiguous results. DEPC-CL/MS can be used to clarify ambiguous HDX/MS results because of its relatively slow (sec-min) intrinsic reaction rate. DEPC-CL/MS does not detect protein dynamics that are measured by HDX/MS. Thus, it can indicate which HDX decreases are a result of loss of solvent exposure and which are a result of a loss of protein dynamics. HDX/MS in parallel with DEPC-CL/MS can provide complementary HOS information about heat stressed monoclonal antibodies (mAbs). Using heat stressed rituximab as a model system, the two methods are used together to provide site-specific information about subtle conformational changes that are undetectable by traditional techniques. DEPC-CL/MS is more sensitive to subtle HOS structural changes occurring at low heat stress while at high heat stress we find that the two methods provide complementary information. The methods are also used to map the epitope in the antigen of a well characterized antibody-antigen system. Using the model system TNFα in complex with a variety of mAbs, we find that DEPC-CL/MS can reveal accurate information about epitopes and subtle structural changes away from epitopes. Moreover, we demonstrate that when HDX/MS is used in parallel with DEPC-CL/MS to study the HOS changes of TNFα upon antibody binding, DEPC-CL/MS can clarify ambiguous HDX results. DEPC-CL/MS can help pinpoint which HDX decreases are due to epitope binding and which decreases are due to TNFα stabilization. Our work highlights the effectiveness of these two methods together to study protein systems that are difficult to study by other methods.

DOI

https://doi.org/10.7275/24371053

Share

COinS