HLA-I Peptide Isolation from Cell Lines

HLA-I peptides were obtained from K562 and HCT116 cells as described previously [33 (link)]. In brief, 1 × 10⁹ cells were dissociated using 40 mL of lysis buffer with 0.25% Sodium deoxycholate, 1% n-octyl glucoside, 100 mM PMSF and protease inhibitor cocktails in PBS at 4 °C for 60 min. Lysate were further cleared by 30 min centrifugation at 14,000× g. Cleared lysate were immunoaffinity purified with pan-HLA class I complexes antibody covalently bound to Protein-A Sepharose CL-4B beads. Beads were first washed with 10 column volumes of 150 mM NaCl, 20 mM Tris HCl (buffer A), then 10 column volumes of 400 mM NaCl, 20 mM Tris HCl, then 10 volumes of buffer A again, and finally with 10 column volumes of 20 mM Tris HCl, pH 8.0. The HLA-I molecules were eluted at room temperature using 0.1 N acetic acid. Eluate were then loaded on Sep-Pak tC18 cartridges (Waters, 50 mg) and washed with 0.1% TFA. The peptides were separated from HLA-I complexes on the C18 cartridges by eluting with 30% ACN in 0.1% TFA and concentrated to 20 µL using vacuum centrifugation. Finally, a 5 µL sample was used for MS analysis.