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.

Metal Ions and Chemical Modification Reagents Inhibit the Enzymatic Activity of Lecithin-Dependent Hemolysin from Vibrio parahaemolyticus.

Lecithin-dependent thermolabile hemolysin (LDH) is a virulence factor excreted by Vibrio parahaemolyticus, a marine bacterium that causes important losses in shrimp farming. In this study, the function of LDH was investigated through its inhibition by metal ions (Mg2+, Ca2+, Mn2+, Co2+, Ni2+ and Cu2+) and chemical modification reagents: ß-mercaptoethanol (ßME), phenylmethylsulfonyl fluoride (PMSF) and diethyl pyrocarbonate (DEPC). LDH was expressed in the Escherichia coli strain BL-21, purified under denaturing conditions, and the enzymatic activity was evaluated. Cu2+, Ni2+, Co2+ and Ca2+ at 1 mmol/L inhibited the LDH esterase activity by 20−95%, while Mg2+ and Mn2+ slightly increased its activity. Additionally, PMSF and DEPC at 1 mmol/L inhibited the enzymatic activity by 40% and 80%, respectively. Dose-response analysis showed that DEPC was the best-evaluated inhibitor (IC50 = 0.082 mmol/L), followed by Cu2+ > Co2+ > Ni2+ and PMSF (IC50 = 0.146−1.5 mmol/L). Multiple sequence alignment of LDH of V. parahaemolyticus against other Vibrio species showed that LDH has well-conserved GDSL and SGNH motifs, characteristic of the hydrolase/esterase superfamily. Additionally, the homology model showed that the conserved catalytic triad His-Ser-Asp was in the LDH active site. Our results showed that the enzymatic activity of LDH from V. parahaemolyticus was modulated by metal ions and chemical modification, which could be related to the interaction with catalytic amino acid residues such as Ser153 and/or His 393.

Accounting for Neighboring Residue Hydrophobicity in Diethylpyrocarbonate Labeling Mass Spectrometry Improves Rosetta Protein Structure Prediction.

Covalent labeling mass spectrometry allows for protein structure elucidation via covalent modification and identification of exposed residues. Diethylpyrocarbonate (DEPC) is a commonly used covalent labeling reagent that provides insight into structure through the labeling of lysine, histidine, serine, threonine, and tyrosine residues. We recently implemented a Rosetta algorithm that used binary DEPC labeling data to improve protein structure prediction efforts. In this work, we improved on our modeling efforts by accounting for the level of hydrophobicity of neighboring residues in the microenvironment of serine, threonine, and tyrosine residues to obtain a more accurate estimate of the hydrophobic neighbor count. This was incorporated into Rosetta functionality, along with considerations for solvent-exposed histidine and lysine residues. Overall, our new Rosetta score term successfully identified best scoring models with less than 2 Å root-mean-squared deviations (RMSDs) for five of the seven benchmark proteins tested. We additionally developed a confidence metric to measure prediction success for situations in which a native structure is unavailable.

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.

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.

Diethylpyrocarbonate Footprints a Membrane Protein in Micelles.

Membrane proteins play crucial roles in cell signaling and transport and, thus, are the targets of many small molecule drugs. The characterization of membrane protein structures poses challenges for the high-resolution biophysical tools because the transmembrane (TM) domain is hydrophobic, opening an opportunity for mass spectrometry (MS)-based footprinting. The hydrophobic reagent diethylpyrocarbonate (DEPC), a heavily studied footprinter for water-soluble proteins, can label up to 30% of surface residues via a straightforward protocol, streamlining the MS-based footprinting workflow. To test its applicability to membrane proteins, we footprinted vitamin K epoxide reductase (VKOR) membrane protein with DEPC. The results demonstrate that besides labeling the hydrophilic extracellular (extramembrane (EM)) domain, DEPC can also diffuse into the hydrophobic TM domain and subsequently label that region. The labeling process was facilitated by tip sonication to enhance reagent diffusion into micelles. We then analyzed the correlation between the residue modification extent and the theoretical accessible surface area percentage (%ASA); the data generally show good correlation with the residue location. Compared with conventional hydrophilic footprinters, the relatively hydrophobic DEPC can map a membrane protein's TM domain, suggesting that the reagent's hydrophobicity can be exploited to obtain structural information on the membrane-spanning region. This encouraging result should assist in the development of more efficient footprinters for membrane protein TM domain footprinting, enabled by further understanding the relationship between a reagent's hydrophobicity and its preferred labeling sites.

Kinetic study of selected microorganisms and quality attributes during cold storage of mango and passion fruit smoothie subjected to dimethyl dicarbonate.

The objectives of this research were to study the effect of DMDC (0-250 ppm) on quality and shelf life of mango and passion fruit smoothie during cold storage. The correlation between microbial population (total microorganisms, yeast and mold, E. coli and S. aureus) and DMDC concentration using zero-order kinetic and first-order kinetic was also determined. In addition, the effect of DMDC compared with pasteurization (90 °C, 100 s) on quality of mixed mango and passion fruit smoothie during the cold storage (4 °C) was studied. The results showed that microbial inactivation was best-described by first-order kinetic model due to a higher coefficient of determination (R2). In addition, DMDC did not affect the decreasing trend of total soluble solid, color difference (∆E*) and total phenolic compound as compared to control during the cold storage. DMDC also hindered the increasing trend in microbial population and prevented the loss of antioxidant activity (DPPH and FRAP assays) and total flavonoid content and decreased the PPO activity as compared with the control during the cold storage. In summary, DMDC showed the potential to maintain the quality and to extend the shelf life of mango and passion fruit smoothie during cold storage.

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.

Utilization of Hydrophobic Microenvironment Sensitivity in Diethylpyrocarbonate Labeling for Protein Structure Prediction.

Diethylpyrocarbonate (DEPC) labeling analyzed with mass spectrometry can provide important insights into higher order protein structures. It has been previously shown that neighboring hydrophobic residues promote a local increase in DEPC concentration such that serine, threonine, and tyrosine residues are more likely to be labeled despite low solvent exposure. In this work, we developed a Rosetta algorithm that used the knowledge of labeled and unlabeled serine, threonine, and tyrosine residues and assessed their local hydrophobic environment to improve protein structure prediction. Additionally, DEPC-labeled histidine and lysine residues with higher relative solvent accessible surface area values (i.e., more exposed) were scored favorably. Application of our score term led to reductions of the root-mean-square deviations (RMSDs) of the lowest scoring models. Additionally, models that scored well tended to have lower RMSDs. A detailed tutorial describing our protocol and required command lines is included. Our work demonstrated the considerable potential of DEPC covalent labeling data to be used for accurate higher order structure determination.

Complementary Structural Information for Stressed Antibodies from Hydrogen-Deuterium Exchange and Covalent Labeling Mass Spectrometry.

Identifying changes in the higher-order structure (HOS) of therapeutic monoclonal antibodies upon storage, stress, or mishandling is important for ensuring efficacy and avoiding adverse effects. Here, we demonstrate diethylpyrocarbonate (DEPC)-based covalent labeling (CL) mass spectrometry (MS) and hydrogen-deuterium exchange (HDX)/MS can be used together to provide site-specific information about subtle conformational changes that are undetectable by traditional techniques. Using heat-stressed rituximab as a model protein, we demonstrate that CL/MS is more sensitive than HDX/MS to subtle HOS structural changes under low stress conditions (e.g., 45 and 55 °C for 4 h). At higher heat stress (65 °C for 4 h), we find CL/MS and HDX/MS provide complementary information, as CL/MS reports on changes in side chain orientation while HDX/MS reveals changes in backbone dynamics. More interestingly, we demonstrate that the two techniques work synergistically to identify likely aggregation sites in the heat-stressed protein. In particular, the CH3 and CL domains experience decreases in deuterium uptake after heat stress, while only the CH3 domain experiences decreases in DEPC labeling extent as well, suggesting the CH3 domain is a likely site of aggregation and the CL domain only undergoes a decrease in backbone dynamics. The combination of DEPC-CL/MS and HDX/MS provides valuable structural information, and the two techniques should be employed together when investigating the HOS of protein therapeutics.

Modified µ-QuEChERS coupled to diethyl carbonate-based liquid microextraction for PAHs determination in coffee, tea, and water prior to GC-MS analysis: An insight to reducing the impact of caffeine on the GC-MS measurement.

A fast, sensitive and eco-friendly method was developed for the determination of fifteen polycyclic aromatic hydrocarbons (PAHs) in different environmental matrices through gas chromatography mass spectrometry (GC-MS). The method utilizes a modified and miniaturized quick easy cheap effective rugged and safe (QuEChERS) clean up procedure coupled to an air-assisted dispersive liquid-liquid microextraction (AA-DLLME) for the enrichment of the concerned compounds. The AA-DLLME uses diethyl carbonate (DEC) as a green bio-based solvent for the microextraction. DEC is considered as biodegradable (with octanol/water coefficient < 3, resulting in low potential of bioaccumulation), classified as a green solvent and considered as one of the recommended solvent alternatives based on SSG results. The AA-DLLME procedure was optimized by One-Variable-at-A-Time (OVAT) succeeded by experimental design applying Central Composite Face-centered (CCF) design. The method linear calibration was found in the range of 10-120 µg/Kg for Benzo[a]pyrene and 5-100 µg/Kg for all other PAHs with low detection limits ranging from 0.01 to 2.10 µg/Kg. It could enrich the PAHs up to 166-folds. The combination of modified µ-QuEChERS with the green AA-DLLME could sharply decrease the caffeine amount on the final extract injected to the GC-MS instrument. The method was successfully applied to coffee, tea, and water samples with acceptable % recovery (>90%). Finally, the impact of our procedure to the environment from green analytical chemistry view was assessed by a novel metric system called AGREE, proving the greenness of our procedure.

Protein-Ligand Affinity Determinations Using Covalent Labeling-Mass Spectrometry.

Determining the binding affinity is an important aspect of characterizing protein-ligand complexes. Here, we describe an approach based on covalent labeling (CL)-mass spectrometry (MS) that can accurately provide protein-ligand dissociation constants (Kd values) using diethylpyrocarbonate (DEPC) as the labeling reagent. Even though DEPC labeling reactions occur on a time scale that is similar to the dissociation/reassociation rates of many protein-ligand complexes, we demonstrate that relatively accurate binding constants can still be obtained as long as the extent of protein labeling is kept below 30%. Using two well-established model systems and one insufficiently characterized system, we find that Kd values can be determined that are close to values obtained in previous measurements. The CL-MS-based strategy that is described here should serve as an alternative for characterizing protein-ligand complexes that are challenging to measure by other methods. Moreover, this method has the potential to provide, simultaneously, the affinity and binding site information.

Covalent Labeling/Mass Spectrometry of Monoclonal Antibodies with Diethylpyrocarbonate: Reaction Kinetics for Ensuring Protein Structural Integrity.

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.

Copper(II) partially protects three histidine residues and the N-terminus of amyloid-ß peptide from diethyl pyrocarbonate (DEPC) modification.

Diethyl pyrocarbonate (DEPC) has been primarily used as a residue-specific modifying agent to study the role of His residues in peptide/protein and enzyme function; however, its action is not specific, and several other residues can also be modified. In the current study, we monitored the reaction of DEPC with amyloid-beta (Aß) peptides and insulin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and determined the modification sites by electrospray ionization quadrupole time-of-flight tandem MS (ESI Q-TOF MS/MS). Our results indicate that five residues in Aß1-42 are modified in the presence of 30-fold molar excess of DEPC. After hydroxylamine treatment (which removes modifications from three His residues), two labels remain bound at the peptide N terminus and Lys16. DEPC treatment of Aß1-42 promotes peptide aggregation, as monitored through the loss of soluble Aß42 in a semi-quantitative MALDI-TOF MS assay. It has been previously proposed that Cu(II) ions protect Aß1-16 from DEPC modification through binding to His6. We confirmed that Cu(II) ions decrease the stoichiometry of Aß1-16 modification with the excess of DEPC being lower as compared to the control, which indicates that Cu(II) protects Aß from DEPC modification. Sequencing of obtained Cu(II)-protected Aß1-16 samples showed that Cu(II) does not protect any residues completely, but partially protects all three His residues and the N terminus. Thus, the protection by Cu(II) ions is not related to specific metal binding to a particular residue (e.g. His6), but rather all His residues and the N terminus are involved in binding of Cu(II) ions. These results allow us to elucidate the effect of DEPC modification on amyloidogenity of human Aß and to speculate about the role of His residues in these processes.

Higher-Order Structure Influences the Kinetics of Diethylpyrocarbonate Covalent Labeling of Proteins.

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.

The combined effect of high pressure processing and dimethyl dicarbonate to inactivate foodborne pathogens in apple juice.

Novel processing technologies can be used to improve both the microbiological safety and quality of food products. The application of high pressure processing (HPP) in combination with dimethyl dicarbonate (DMDC) represents a promising alternative to classical thermal technologies. This research work was undertaken to investigate the combined effect of HPP and DMDC, which was aimed at reaching over 5-log reduction in the reference pathogens Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes inoculated in apple juice. Different strains of each species were tested. The pressure (ranging from 100 to 600 MPa), dwell time (from 26 to 194 s), and DMDC (from 116 to 250 mg/L) were tested based on a central composite rotatable design. The dwell time, in the studied range, did not have a significant effect (p > 0.1) on the pathogens´ reduction. All treatments achieved a greater than 5-log reduction for E. coli O157:H7 and L. monocytogenes. The reductions for S. enterica were also greater than 5-log for almost all tested combinations. The results for S. enterica suggested that it is more resistant to HPP and DMDC compared with E. coli O157:H7 and L. monocytogenes. The findings of this study showed that DMDC at low concentrations can be added to apple juice to reduce the parameters conventionally applied in HPP. The combined use of HPP and DMDC was highly effective under the conditions of this study.

Synergistic Structural Information from Covalent Labeling and Hydrogen-Deuterium Exchange Mass Spectrometry for Protein-Ligand Interactions.

Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) and covalent labeling (CL) MS are typically considered to be complementary methods for protein structural analysis, because one probes the protein backbone, while the other probes side chains. For protein-ligand interactions, we demonstrate in this work that the two labeling techniques can provide synergistic structural information about protein-ligand binding when reagents like diethylpyrocarbonate (DEPC) are used for CL because of the differences in the reaction rates of DEPC and HDX. Using three model protein-ligand systems, we show that the slower time scale for DEPC labeling makes it only sensitive to changes in solvent accessibility and insensitive to changes in protein structural fluctuations, whereas HDX is sensitive to changes in both solvent accessibility and structural fluctuations. When used together, the two methods more clearly reveal binding sites and ligand-induced changes to structural fluctuations that are distant from the binding site, which is more comprehensive information than either technique alone can provide. We predict that these two methods will find widespread usage together for more deeply understanding protein-ligand interactions.

Regioselective Single-Electron Tsuji-Trost Reaction of Allylic Alcohols: A Photoredox/Nickel Dual Catalytic Approach.

A radical-mediated functionalization of allyl alcohol derived partners with a variety of alkyl 1,4-dihydropyridines via photoredox/nickel dual catalysis is described. This transformation transpires with high linear and E-selectivity, avoiding the requirement of harsh conditions (e.g., strong base, elevated temperature). Additionally, using aryl sulfinate salts as radical precursors, allyl sulfones can also be obtained. Kinetic isotope effect experiments implicated oxidative addition of the nickel catalyst to the allylic electrophile as the turnover-limiting step, supporting previous computational studies.

Covalent Labeling with Diethylpyrocarbonate: Sensitive to the Residue Microenvironment, Providing Improved Analysis of Protein Higher Order Structure by Mass Spectrometry.

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.