Fig. 1. Platform for quantitative immunopeptidomics using hipMHCs.

a HipMHCs were generated through UV-mediated peptide exchange of HLA*A2:01 monomers with a heavy leucine HLA-A*02:01 binding peptide. Stable hipMHCs concentrations were measured with an ELISA, and hipMHC complexes were added to lysate samples prior to immunoprecipitation (IP), at the same concentration for quantification correction (blue/teal) or titrated in to create an internal standard curve (red). Heavy and light pMHCs were isolated with IP, acid elution, and molecular weight cut-off (MWCO) filters. b Peptides were analyzed by LC-MS/MS three ways. Relative quantification label-free analyses were quantified by integrating the area under the curve (AUC) of the chromatographic elution across samples, and quantification was normalized by applying correction factors determined by hipMHC AUC intensity ratios between samples. Samples for multiplexed analysis were TMT-labeled and relative quantification was implemented using reporter ion intensities. Normalization was performed using hipMHC reporter ion intensity ratios across TMT channels. For absolute quantification, TMT-labeled samples containing a hipMHC internal standard curve were used to calculate the endogenous copies per cell of the pMHC of interest.