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Author ORCID Identifier

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program


Year Degree Awarded


Month Degree Awarded


First Advisor

Richard W. Vachet

Subject Categories

Amino Acids, Peptides, and Proteins | Analytical Chemistry | Other Biochemistry, Biophysics, and Structural Biology | Other Pharmacy and Pharmaceutical Sciences


Using mass spectrometry (MS) to obtain information about a higher order structure of protein requires that a protein’s structural properties are encoded into the mass of that protein. Covalent labeling (CL) with reagents that can irreversibly modify solvent accessible amino acid side chains is an effective way to encode structural information into the mass of a protein, as this information can be read-out in a straightforward manner using standard MS-based proteomics techniques. The differential reactivity of proteins under two or more conditions can be used to distinguish protein topologies, conformations, and/or binding sites. CL-MS methods have been effectively used for the structural analysis of proteins and protein therapeutics.

This dissertation focuses on the use of a diethylpyrocarbonate (DEPC-based CL-MS method to characterize the higher-order structure of protein therapeutics. DEPC is a simple to use, commercially-available covalent labeling reagent that can readily react with a range of nucleophilic residues in proteins. We find that in intact proteins weakly nucleophilic side chains (Ser, Thr, and Tyr) can be modified by DEPC in addition to other residues such as His, Lys, and Cys, providing very good structural resolution. We hypothesize that the microenvironment around these side chains, as formed by a protein’s higher order structure, tunes their reactivity such that they can be labeled. To test this hypothesis, we compare DEPC labeling reactivity of Ser, Thr, and Tyr residues in intact proteins with peptide fragments from the same proteins. Results indicate that these residues almost never react with DEPC in free peptides, supporting the hypothesis that a protein’s local microenvironment tunes the reactivity of these residues. From a close examination of the structural features near the reactive residues, we find that nearby hydrophobic residues are essential, suggesting that the enhanced reactivity of certain Ser, Thr, and Tyr residues occurs due to higher local concentrations of DEPC.

Monoclonal antibodies (mAbs) are among the fastest growing therapeutics in the pharmaceutical industry. Detecting higher-order structure changes of antibodies upon storage or mishandling, however, is a challenging problem. In this dissertation, we describe the use of DEPC-based CL-MS to detect conformational changes caused by heat stress, using rituximab as a model system. The structural resolution obtained from DEPC CL-MS is high enough to probe subtle conformation changes that are not detectable by common biophysical techniques. Results demonstrate that DEPC CL-MS can detect and identify sites of conformational changes at the temperatures below the antibody melting temperature (e.g., 55 ᴼC). The observed labeling changes at lower temperatures are validated by activity assays that indicate changes in the Fab region. At higher temperatures (e.g., 65 ᴼC), conformational changes and aggregation sites are identified from changes in CL levels, and these results are confirmed by complementary biophysical and activity measurements. Given the sensitivity and simplicity of DEPC CL-MS, this method should be amenable to the structural investigations of other antibody therapeutics.

Reliable information about antibody higher-order structure can be obtained, though, only when the protein’s structural integrity is preserved during labeling. In this dissertation, we have evaluated the applicability of DEPC reaction kinetics for ensuring the structural integrity of mAbs during labeling. By monitoring the modification extent of selected proteolytic fragments as a function of DEPC concentration, we find that a common DEPC concentration can be used for different monoclonal antibodies in formulated samples without perturbing their higher-order structure. Under these labeling conditions, we find that the antibodies can accommodate up to four DEPC modifications without being structurally perturbed, indicating that multi-domain proteins can withstand more than one label, which contrasts to previously studied single-domain proteins. This more extensive labeling provides a more sensitive measure of structure, making DEPC-based CL-MS suitable for the higher-order structural analyses of mAbs.