Dataset Information

Epitope Mapping with Diethylpyrocarbonate Covalent Labeling-Mass Spectrometry.

ABSTRACT: 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.

SUBMITTER: Tremblay CY

PROVIDER: S-EPMC8785103 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Epitope Mapping with Diethylpyrocarbonate Covalent Labeling-Mass Spectrometry.

Tremblay Catherine Y CY Kirsch Zachary J ZJ Vachet Richard W RW

Analytical chemistry 20211221 2

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, threon ...[more]

PMID: 34932327

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Project description:Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for ?-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for cytochrome c, and 9 to 8 Å for ?-2-microglobulin.

Dataset Information

Epitope Mapping with Diethylpyrocarbonate Covalent Labeling-Mass Spectrometry.

Publications

Epitope Mapping with Diethylpyrocarbonate Covalent Labeling-Mass Spectrometry.

Similar Datasets

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