HLA-I Peptide Elution and Identification

Peptides were eluted from immunoprecipitated HLA-I molecules as described previously (82 (link)). In brief, transduced or untransduced H1975 cell lines were expanded in 10 cm plates (Corning, 430599), lysed using Triton X-100 (Sigma Aldrich, T9284), and incubated overnight at 4°C under gentle agitation with 50 μg of the pan HLA-A, B, C-specific antibody W6/32 for every 10 mg of protein. HLA class I molecules were immunoprecipitated by using Protein A/G UltraLink resin beads (ThermoFisher Scientific, 53133) and then directly eluted on columns (ThermoFisher Scientific, 89897) using 0.1 N acetic acid (Fisher Scientific, BP2401). HLA-I purification was confirmed by Western blot and eluted fractions were pooled prior to LC-MS/MS analysis.