Proteins Can Withstand More Extensive Labeling while Providing Accurate Structural Information in Covalent Labeling-Mass Spectrometry.

Diethylpyrocarbonate (DEPC) covalent labeling-mass spectrometry (CL-MS) has been extensively utilized to study protein structure and interactions owing to its ease of use, commercial availability, and broad labeling of nucleophilic residues. During typical CL-MS experiments with DEPC, the extent of labeling is kept low to avoid any structural perturbations resulting from covalent modification of the protein. In this study, we demonstrate that proteins can be labeled more extensively via DEPC and still provide accurate structural information. To show this, we modeled labeling kinetics over a range of DEPC concentrations and used molecular dynamics simulations to investigate the molecular-level effects of extensive labeling on the protein structure. Our results indicate that higher extents of DEPC labeling do not significantly perturb the protein structure and can lead to improved precision, detectability of labeled peptides, and protein structural resolution. Furthermore, higher extents of labeling enable better identification of protein-ligand binding sites where lower extents of modification provide ambiguous results.

Multisite Labeling of Proteins Using the Ligand-Directed Reactivity of Triggerable Michael Acceptors.

Targeted modification of endogenous proteins without genetic manipulation of protein expression machinery has a range of applications from chemical biology to drug discovery. Despite being demonstrated to be effective in various applications, target-specific protein labeling using ligand-directed strategies is limited by stringent amino acid selectivity. Here, we present highly reactive ligand-directed triggerable Michael acceptors (LD-TMAcs) that feature rapid protein labeling. Unlike previous approaches, the unique reactivity of LD-TMAcs enables multiple modifications on a single target protein, effectively mapping the ligand binding site. This capability is attributed to the tunable reactivity of TMAcs that enable the labeling of several amino acid functionalities via a binding-induced increase in local concentration while remaining fully dormant in the absence of protein binding. We demonstrate the target selectivity of these molecules in cell lysates using carbonic anhydrase as the model protein. Furthermore, we demonstrate the utility of this method by selectively labeling membrane-bound carbonic anhydrase XII in live cells. We envision that the unique features of LD-TMAcs will find use in target identification, investigation of binding/allosteric sites, and studying membrane proteins.

Membrane Protein Binding Interactions Studied in Live Cells via Diethylpyrocarbonate Covalent Labeling Mass Spectrometry.

Membrane proteins are vital in the human proteome for their cellular functions and make up a majority of drug targets in the U.S. However, characterizing their higher-order structures and interactions remains challenging. Most often membrane proteins are studied in artificial membranes, but such artificial systems do not fully account for the diversity of components present in cell membranes. In this study, we demonstrate that diethylpyrocarbonate (DEPC) covalent labeling mass spectrometry can provide binding site information for membrane proteins in living cells using membrane-bound tumor necrosis factor α (mTNFα) as a model system. Using three therapeutic monoclonal antibodies that bind TNFα, our results show that residues that are buried in the epitope upon antibody binding generally decrease in DEPC labeling extent. Additionally, serine, threonine, and tyrosine residues on the periphery of the epitope increase in labeling upon antibody binding because of a more hydrophobic microenvironment that is created. We also observe changes in labeling away from the epitope, indicating changes to the packing of the mTNFα homotrimer, compaction of the mTNFα trimer against the cell membrane, and/or previously uncharacterized allosteric changes upon antibody binding. Overall, DEPC-based covalent labeling mass spectrometry offers an effective means of characterizing structure and interactions of membrane proteins in living cells.

Diethylpyrocarbonate-Based Covalent Labeling Mass Spectrometry of Protein Interactions in a Membrane Complex System.

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.

Complementary Structural Information for Antibody-Antigen Complexes from Hydrogen-Deuterium Exchange and Covalent Labeling Mass Spectrometry.

Characterizing antibody-antigen interactions is necessary for properly developing therapeutic antibodies, understanding their mechanisms of action, and patenting new drug molecules. Here, we demonstrate that hydrogen-deuterium exchange (HDX) mass spectrometry (MS) measurements together with diethylpyrocarbonate (DEPC) covalent labeling (CL) MS measurements provide higher order structural information about antibody-antigen interactions that is not available from either technique alone. Using the well-characterized model system of tumor necrosis factor α (TNFα) in complex with three different monoclonal antibodies (mAbs), we show that two techniques offer a more complete overall picture of TNFα's structural changes upon binding different mAbs, sometimes providing synergistic information about binding sites and changes in protein dynamics upon binding. Labeling decreases in CL generally occur near the TNFα epitope, whereas decreases in HDX can span the entire protein due to substantial stabilization that occurs when mAbs bind TNFα. Considering both data sets together clarifies the TNFα regions that undergo a decrease in solvent exposure due to mAb binding and that undergo a change in dynamics due to mAb binding. Moreover, the single-residue level resolution of DEPC-CL/MS can clarify HDX/MS data for long peptides. We feel that the two techniques should be used together when studying the mAb-antigen interactions because of the complementary information they provide.

Covalent Labeling-Mass Spectrometry Provides a Molecular Understanding of Noncovalent Polymer-Protein Complexation.

The delivery of functional proteins to the intracellular space offers tremendous advantages for the development of new therapeutics but is limited by the passage of these large polar biomacromolecules through the cell membrane. Noncovalent polymer-protein binding that is driven by strong carrier-cargo interactions, including electrostatics and hydrophobicity, has previously been explored in the context of delivery of functional proteins. Appropriately designed polymer-based carriers can take advantage of the heterogeneous surface of protein cargoes, where multiple types of physical binding interactions with polymers can occur. Traditional methods of assessing polymer-protein binding, including dynamic light scattering, circular dichroism spectroscopy, and fluorescence-based assays, are useful in the study of new polymer-based carriers but face a number of limitations. We implement for the first time the method of covalent labeling-mass spectrometry (CL-MS) to probe intermolecular surface interactions within noncovalent polymer-protein complexes. We demonstrate the utility of CL-MS for establishing binding of an amphiphilic block copolymer to negatively charged and hydrophobic surface patches of a model protein, superfolder green fluorescent protein (sfGFP), using diethylpyrocarbonate as a pseudo-specific labeling reagent. In addition, we utilize this method to explore differences at the intermolecular surface as the ratio of polymer to protein increases, particularly in the context of defining effective protein delivery regimes. By promoting an understanding of the intermolecular interactions in polymer-protein binding and identifying sites where polymers bind to protein surfaces, noncovalent polymer carriers can be more effectively designed for protein delivery applications.

Distinguishing Histidine Tautomers in Proteins Using Covalent Labeling-Mass Spectrometry.

In this work, we use diethylpyrocarbonate (DEPC)-based covalent labeling together with LC-MS/MS analysis to distinguish the two sidechain tautomers of histidine residues in peptides and proteins. From labeling experiments on model peptides, we demonstrate that DEPC reacts equally with both tautomeric forms to produce chemically different products with distinct dissociation patterns and LC retention times, allowing the ratios of the two tautomers to be determined in peptides and proteins. Upon measuring the tautomer ratios of several histidine residues in myoglobin, we find good agreement with previous 2D NMR data on this protein. Because our DEPC labeling/MS approach is simpler, faster, and more precise than 2D NMR, our method will be a valuable way to determine how protein structure enforces histidine sidechain tautomerization. Because the tautomeric state of histidine residues is often important for protein structure and function, the ability of DEPC labeling/MS to distinguish histidine tautomers should equip researchers with a tool to understand the histidine residue structure and function more deeply in proteins.

Epitope Mapping with Diethylpyrocarbonate Covalent Labeling-Mass Spectrometry.

Antigen-antibody epitope mapping is essential for understanding binding mechanisms and developing new protein therapeutics. In this study, we investigate diethylpyrocarbonate (DEPC) covalent labeling-mass spectrometry as a means of analyzing antigen-antibody interactions using the well-characterized model system of TNFα in complex with three different antibodies. Results show that residues buried in the epitope undergo substantial decreases in labeling, as expected. Interestingly, serine, threonine, and tyrosine residues at the edges of the epitope undergo unexpected increases in labeling. The increased labeling of these weakly nucleophilic residues is caused by the formation of hydrophobic pockets upon antibody binding that presumably increase local DEPC concentrations. Residues that are distant from the epitope generally do not undergo changes in labeling extent; however, some that do change experience variations in their local microenvironment due to side-chain reorganization or stabilization of the TNFα trimer that occurs upon binding. Overall, DEPC labeling of antigen-antibody complexes is found to depend on both changes in solvent exposure and changes to the residue microenvironment.

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry.

Characterizing a protein's higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein's structure by MS, specific chemical reactions are performed in solution that encode a protein's structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as ß2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.