Characterization of HLA-I Peptide Complexes

C1R were infected at MOI5 for 12hrs and snap frozen as previously^{26 (link)}. HLA class I complexes were isolated as described^{31 (link)}. Briefly, C1R cell pellets were disrupted by cryogenic milling (Retsch Mixer Mill MM 400) and lysed with 0.5% IGEPAL CA-630, 50 mM Tris-HCl pH 8.0, 150 mM NaCl and protease inhibitors (cOmplete Protease Inhibitor Cocktail Tablet; Roche Molecular Biochemicals) for 1 h at 4 °C with rolling. Lysates were cleared by ultracentrifugation and the HLA class I complexes isolated by immunoaffinity purification using pan-HLA class I antibody (W6/32) bound to protein-A-agarose. Peptide-HLA were dissociated with 10% acetic acid and fractionated by reversed-phase high-performance liquid chromatography. Fractions containing peptides were combined into 9–10 pools, vacuum-concentrated, then reconstituted in 15 µl 0.1% formic acid in Optima™ LC-MS water with indexed retention time (iRT) peptides^{77 (link)}. Peptides were analyzed by LC-MS/MS on a Q-Exactive Plus Hybrid Quadrupole Orbitrap (Thermo Fisher Scientific) as described^{24 (link),25 (link)}.