Fig. 5: Mapping reactive cysteines on MHC-I-bound antigens using the single-chain trimer model.

a Schematic representation of a native pMHC-I complex and a single-chain trimer. b Constructs of SCTs of HLA-A*02:01 presenting the KRAS-G12C and KRAS-G12D neoantigens. SCT, single-chain trimer. LTR, long-terminal repeats. CMV, cytomegalovirus. SP, signal peptide. c In-gel fluorescence analysis of KRAS-G12C-SCT and KRAS-G12D-SCT from HEK293T cells treated with the MSD probe (50 µM, 30 min). The result is a representative of three experiments (n = 3 independent replicates) TAMRA, tetramethylrhodamine. d ELISA assay measuring the assembly of MHC-I and β2-microglobulin in the presence of sulfonated maleimide probes. Data represent mean values ± SEM (n = 3 independent replicates). e Modeling studies suggest that the cysteine in the KRAS-G12C neoantigen is exposed to the solvent when bound to MHC-I. f In-gel fluorescence analysis of SCTs of HLA-A*02:01 presenting pp65 antigens with cysteines introduced at positions 1–9 from HEK293T cells treated with the MSD probe (50 µM, 30 min). The bar graph represents quantification of the TAMRA/FLAG ratio values following FLAG immunoprecipitation (IP). Data represent mean values (n = 2 independent replicates). Source data are provided as a Source Data file.