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Author ORCID Identifier


Open Access Dissertation

Document Type


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 | Biochemistry


The study of protein higher-order structures is vital because it is closely related to the investigation of protein folding, aggregation, interaction and protein therapeutics. Consequently, numerous biochemical and biophysical tools have been developed to study protein higher-order structures in many different situations. The combination of covalent labeling (CL) and mass spectrometry (MS) has emerged as a powerful tool for studying protein structures and offers many advantages over other traditional techniques, such as better structural coverage, high throughput, high sensitivity, and the ability to study proteins in mixtures. This dissertation focuses on diethylpyrocarbonate (DEPC) as an effective CL reagent that can label N-termini and the side chains of several nucleophilic residues, providing information for about 30% of the residues in the average protein. In contrast to hydroxyl radical foot printing, it does not require expensive laser apparatus. The reaction only generates a single type of product, which facilitates the data interpretation. The overarching goal of this dissertation is to continue the development and application of DEPC-based CL-MS studies of protein structure and assembly. Specifically, the first part of my dissertation concentrates on the kinetic study of DEPC labeling and the second part focuses its applications to a set of biologically important systems. We first established a quantitative correlation between general protein structural factors and DEPC reaction rates. Our results show that the solvent accessible surface areas of histidine and lysine residues in proteins are the primary factors that determine a protein’s reactivity towards DEPC. This model can be used to predict the reactivity of a protein based on its structure and sequence, allowing the optimal DEPC concentration to be chosen for a given protein. In addition, we determined that the overall reaction of DEPC with peptides was second order and measured the intrinsic rate coefficients of nucleophilic residues and the N-terminus from the dose-response plots. The histidine reactivity in peptides is primarily impacted by the number of charged residues and peptide net charge, while histidine reactivity in proteins is weakly correlated with their SASA, indicating more complicated factors control their reactivity. Moreover, we use DEPC labeling together with LC-MS/MS analysis to distinguish the two sidechain tautomers of histidine residues in peptides by distinct dissociation patterns and LC retention times. The tautomer ratios of several histidine residues in myoglobin are in good agreement with previous 2D nuclear magnetic resonance (NMR) data. The ability of DEPC labeling/MS to determine histidine tautomeric state will help us to better understand histidine residue structure and function in proteins. Furthermore, we applied CL-MS to study the structures of membrane-associated proteins, an important class of proteins that are difficult to investigate by other techniques. We chose chemotaxis A (CheA) as a model system to study the structural and binding interactions of this protein in a membrane-associated chemoreceptor complex system. The DEPC labeling changes of 20 residues in the complex were monitored. While the results for 17 of these sites are consistent with the current structural model of the chemoreceptor complex, the labeling results for several sites are inconsistent with the model, suggesting that the model may need to be further refined. Last but not the least, we demonstrated that DEPC CL-MS could probe surface interactions in polymer-protein complexes and provided residue-level structural information for polymeric protein transduction domain mimics (PTDM)-protein interactions for the first time. In particular, our results show that, with the increase of polymer concentrations, residues near negatively-charged and hydrophobic patches on superfolder green fluorescent protein decrease in labeling, which is consistent with a model in which PTDM binding is mediated via electrostatic and hydrophobic interactions on the protein surface.