Browse
Submit Data
Databases
API
Help

Dataset Information

23 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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 (K_d 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 K_d 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.

SUBMITTER: Liu T

PROVIDER: S-EPMC7332385 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

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

Project description: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.

| S-EPMC6891134 | biostudies-literature

Using Covalent Labeling and Mass Spectrometry To Study Protein Binding Sites of Amyloid Inhibiting Molecules.

Project description:Amyloid aggregates are associated with several debilitating diseases, and there are numerous efforts to develop small molecule treatments against these diseases. One challenge associated with these efforts is determining protein binding site information for potential therapeutics because amyloid-forming proteins rapidly form oligomers and aggregates, making traditional protein structural analysis techniques challenging. Using ?-2-microglobulin (?2m) as a model amyloid-forming protein along with two recently identified small molecule amyloid inhibitors (i.e., rifamycin SV and doxycycline), we demonstrate that covalent labeling and mass spectrometry (MS) can be used to map small-molecule binding sites for a rapidly aggregating protein. Specifically, three different covalent labeling reagents, namely diethylpyrocarbonate, 2,3-butanedione, and the reagent pair EDC/GEE, are used together to pinpoint the binding sites of rifamycin SV, doxycycline, and another molecule, suramin, which binds but does not inhibit Cu(II)-induced ?2m amyloid formation. The labeling results reveal binding sites that are consistent with the known effects of these molecules on ?2m amyloid formation and are in general agreement with molecular docking results. We expect that this combined covalent labeling approach will be applicable to other protein/small molecule systems that are difficult to study by traditional means.

| S-EPMC5677544 | biostudies-literature

Mapping low-affinity/high-specificity peptide-protein interactions using ligand-footprinting mass spectrometry.

Project description:Short linear peptide motifs that are intracellular ligands of folded proteins are a modular, incompletely understood molecular interaction language in signaling systems. Such motifs, which frequently occur in intrinsically disordered protein regions, often bind partner proteins with modest affinity and are difficult to study with conventional structural biology methods. We developed LiF-MS (ligand-footprinting mass spectrometry), a method to map peptide binding sites on folded protein domains that allows consideration of their dynamic disorder, and used it to analyze a set of D-motif peptide-mitogen-activated protein kinase (MAPK) associations to validate the approach and define unknown binding structures. LiF-MS peptide ligands carry a short-lived, indiscriminately reactive cleavable crosslinker that marks contacts close to ligand binding sites with high specificity. Each marked amino acid provides an independent constraint for a set of directed peptide-protein docking simulations, which are analyzed by agglomerative hierarchical clustering. We found that LiF-MS provides accurate ab initio identification of ligand binding surfaces and a view of potential binding ensembles of a set of D-motif peptide-MAPK associations. Our analysis provides an MKK4-JNK1 structural model, which has thus far been crystallographically unattainable, a potential alternate binding mode for part of the NFAT4-JNK interaction, and evidence of bidirectional association of MKK4 peptide with ERK2. Overall, we find that LiF-MS is an effective noncrystallographic way to understand how short linear motifs associate with specific sites on folded protein domains at the level of individual amino acids.

| S-EPMC6800362 | biostudies-literature

Probing protein structure by amino acid-specific covalent labeling and mass spectrometry.

Project description:For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies.

| S-EPMC2768138 | biostudies-literature

Utility of Covalent Labeling Mass Spectrometry Data in Protein Structure Prediction with Rosetta.

Project description:Covalent labeling mass spectrometry experiments are growing in popularity and provide important information regarding protein structure. Information obtained from these experiments correlates with residue solvent exposure within the protein in solution. However, it is impossible to determine protein structure from covalent labeling data alone. Incorporation of sparse covalent labeling data into the protein structure prediction software Rosetta has been shown to improve protein tertiary structure prediction. Here, covalent labeling techniques were analyzed computationally to provide insight into what labeling data is needed to optimize tertiary protein structure prediction in Rosetta. We have successfully implemented a new scoring functionality that provides improved predictions. We developed two new covalent labeling based score terms that use a "cone"-based neighbor count to quantify the relative solvent exposure of each amino acid. To test our method, we used a set of 20 proteins with structures deposited in the Protein Data Bank. Decoy model sets were generated for each of these 20 proteins, and the normalized covalent labeling score versus RMSD distributions were evaluated. On the basis of these distributions, we have determined an optimal subset of residues to use when performing covalent labeling experiments in order to maximize the structure prediction capabilities of the covalent labeling data. We also investigated how much false negative and false positive data can be tolerated without meaningfully impacting protein structure prediction. Using these new covalent labeling score terms, protein models were rescored and the resulting models improved by 3.9 Å RMSD on average. New models were also generated using Rosetta's AbinitioRelax program under the guidance of covalent labeling information, and improvement in model quality was observed.

| S-EPMC6520167 | biostudies-literature

Covalent labeling-mass spectrometry with non-specific reagents for studying protein structure and interactions.

Project description: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 complexes, particularly for systems that are difficult to study by other more traditional biochemical techniques. This review provides an overview of the non-specific CL approaches that have been combined with MS with a particular emphasis on the reagents that are commonly used, including hydroxyl radicals, carbenes, and diethylpyrocarbonate. We describe the reagent and protein factors that affect the reactivity of amino acid side chains. We also include details about experimental design and workflow, data analysis, recent applications, and some future prospects of CL-MS methods.

| S-EPMC6051898 | biostudies-literature

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

Project description: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.

| S-EPMC7370534 | biostudies-literature

Covalent Labeling with an α,β-Unsaturated Carbonyl Scaffold for Studying Protein Structure and Interactions by Mass Spectrometry.

Project description:A new covalent labeling (CL) reagent based on an α,β-unsaturated carbonyl scaffold has been developed for studying protein structure and protein-protein interactions when coupled with mass spectrometry. We show that this new reagent scaffold can react with up to 13 different types of residues on protein surfaces, thereby providing excellent structural resolution. To illustrate the value of this reagent scaffold, it is used to identify the residues involved in the protein-protein interface that is formed upon Zn(II) binding to the protein β-2-microglobulin. The modular design of the α,β-unsaturated carbonyl scaffold allows facile variation of the functional groups, enabling labeling kinetics and selectivity to be tuned. Moreover, by introducing isotopically enriched functional groups into the reagent structure, labeling sites can be more easily identified by MS and MS/MS. Overall, this reagent scaffold should be a valuable CL reagent for protein higher order structure characterization by MS.

| S-EPMC7207043 | biostudies-literature

Covalent labeling and mass spectrometry reveal subtle higher order structural changes for antibody therapeutics.

Project description:Monoclonal antibodies 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 study, we describe the use of diethylpyrocarbonate (DEPC)-based covalent labeling (CL) - mass spectrometry (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.

| S-EPMC6512938 | biostudies-literature

Oxidation-induced conformational changes in calcineurin determined by covalent labeling and tandem mass spectrometry.

Project description:The Ca(2+)/calmodulin activated phosphatase, calcineurin, is inactivated by H2O2 or superoxide-induced oxidation, both in vivo and in vitro. However, the potential for global and/or local conformation changes occurring within calcineurin as a function of oxidative modification, that may play a role in the inactivation process, has not been examined. Here, the susceptibility of calcineurin methionine residues toward H2O2-induced oxidation were determined using a multienzyme digestion strategy coupled with capillary HPLC-electrospray ionization mass spectrometry and tandem mass spectrometry analysis. Then, regions within the protein complex that underwent significant conformational perturbation upon oxidative modification were identified by monitoring changes in the modification rates of accessible lysine residues between native and oxidized forms of calcineurin, using an amine-specific covalent labeling reagent, S,S'-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), and tandem mass spectrometry. Importantly, methionine residues found to be highly susceptible toward oxidation, and the lysine residues exhibiting large increases in accessibility upon oxidation, were all located in calcineurin functional domains involved in Ca(2+)/CaM binding regulated calcineurin stimulation. These findings therefore provide initial support for the novel mechanistic hypothesis that oxidation-induced global and/or local conformational changes within calcineurin contribute to inactivation via (i) impairing the interaction between calcineurin A and calcineurin B, (ii) altering the low-affinity Ca(2+) binding site in calcineurin B, (iii) inhibiting calmodulin binding to calcineurin A, and/or (iv) by altering the affinity between the calcineurin A autoinhibitory domain and the catalytic center.

| S-EPMC4222536 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data