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 TNFa 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 TNFa 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.

Automated summary

In this study, DEPC covalent labeling-mass spectrometry was used to analyze antigen-antibody interactions, revealing that residues buried in the epitope show decreased labeling while residues at the edges of the epitope show increased labeling. The increased labeling of certain residues is caused by the formation of hydrophobic pockets upon antibody binding. Overall, changes in solvent exposure and residue microenvironment play a significant role in DEPC labeling of antigen-antibody complexes.