Mass spectrometry-based mapping of conformational epitopes on SARS-CoV-2 antigens targeted by monoclonal antibodies

The spike glycoprotein of SARS-CoV-2, particularly its receptor-binding domain (RBD), is a key target for therapeutic monoclonal antibodies (mAbs). Epitope mapping is therefore critical for the development of effective antiviral therapeutics. In this study, diethylpyrocarbonate covalent labeling mass spectrometry (DEPC CL-MS) was applied to map epitopes on the beta (B.1.351) and omicron (B.1.1.529) variants of the SARS-CoV-2 RBD, as well as on the original SARS-CoV-2 spike S₁ subunit, in complex with anti-SARS-CoV-2 mAbs. Combined with bottom-up LC-MS/MS, DEPC labeling enabled site-specific identification of residues exhibiting significant modification changes. Clustering of these residues on the protein surface identified potential epitopes for the 1D1 mAb on the RBDs and for the 1D3 mAb on the C-terminal domain of the S₁ subunit. Structural interpretation was supported by available experimental data, with AlphaFold models providing supplementary context where needed. Thus, "theory guides, but experiment decides": AlphaFold aided epitope identification when high-resolution antigen-antibody complex structures were unavailable, but conclusions were ultimately resolved experimentally. Together, these findings establish DEPC CL-MS as a useful and complementary approach for epitope mapping, providing residue-level insights into antigen-antibody interactions and advancing structural understanding for antiviral mAb development.