Covalent labeling with mass spectrometry is increasingly being used for the structural analysis of proteins. Diethylpyrocarbonate (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.

The combination of covalent labeling (CL) and mass spectrometry (MS) has emerged as a useful tool for studying protein structure due to its good structural coverage, the ability to study proteins in mixtures, and its high sensitivity. Diethylpyrocarbonate (DEPC) is 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. For DEPC to provide accurate structural information, the extent of labeling must be controlled to minimize label-induced structural perturbations. In this work, we establish a quantitative correlation between general protein structural factors and DEPC reaction rates by measuring the reaction rate coefficients for several model proteins. Using principal component and regression analyses, we find that the solvent accessible surface areas of histidine and lysine residues in proteins are the primary factors that determine a protein’s reactivity toward DEPC, despite the fact that other more abundant residues, such as tyrosine, threonine, and serine, are also labeled by DEPC. From the statistical analysis, a model emerges that 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. The resulting model is supported by cross-validation studies and by accurately predicting of the reactivity of five test proteins. Overall, our model reveals interesting insight into the reactivity of proteins with DEPC, and it will facilitate identification of optimal DEPC labeling conditions for proteins.

Diethylpyrocarbonate (DEPC)-based covalent labeling together with mass spectrometry is a promising tool for the higher-order structural analysis of 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 work, we have evaluated the applicability of DEPC reaction kinetics for ensuring the structural integrity of monoclonal antibodies (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 multidomain 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 covalent labeling-mass spectrometry suitable for the higher-order structural analyses of mAbs.

Membrane-associated proteins are important because they mediate interactions between a cell’s external and internal environment and they are often targets of therapeutics. Characterizing their structures and binding interactions, however, is challenging because they typically must be solubilized using artificial membrane systems that can make measurements difficult. Mass spectrometry (MS) is emerging as a valuable tool for studying membrane-associated proteins, and covalent labeling MS has unique potential to provide higher order structure and binding information for these proteins in complicated membrane systems. Here, we demonstrate that diethylpyrocarbonate (DEPC) can be effectively used as a labeling reagent to characterize the binding interactions between a membrane-associated protein and its binding partners in an artificial membrane system. Using chemotaxis histidine kinase (CheA) as a model system, we demonstrate that DEPC-based covalent labeling MS can provide structural and binding information about the ternary complex of CheA with two other proteins that is consistent with structural models of this membrane-associated chemoreceptor system. Despite the moderate hydrophobicity of DEPC, we find that its reactivity with proteins is not substantially influenced by the presence of the artificial membranes. However, correct structural information for this multiprotein chemoreceptor system requires measurements of DEPC labeling at multiple reagent concentrations to enable an accurate comparison between CheA and its ternary complex in the chemoreceptor system. In addition to providing structural information that is consistent with the model of this complex system, the labeling data supplements structural information that is not sufficiently refined in the chemoreceptor model.

Membrane proteins are incredibly important biomolecules because they mediate interactions between a cell's external and internal environment. Obtaining information about membrane protein structure and interactions is thus important for understanding these essential biomolecules. Compared with the analyses of water-soluble proteins, the structural analysis of membrane proteins is more challenging owing to their unique chemical properties and the presence of lipid components that are necessary to solubilize them. The combination of covalent labeling (CL) and mass spectrometry (MS) has recently been applied with great success to study membrane protein structure and interactions. These studies have demonstrated the many advantages that CL-MS methods have over other traditional biophysical techniques. In this review, we discuss both amino acid-specific and non-specific labeling approaches and the special considerations needed to address the unique challenges associated with interrogating membrane proteins. This review highlights the aspects of this approach that require special care to be applied correctly and provides a comprehensive review of the membrane protein systems that have been studied by CL-MS.