Multiplexed MHC Peptidome Analysis

Peptide-bound MHC and phosphopeptide samples were analyzed as previously described (25 (link)). GL261-luc2 and CT2A-luc tumors were flash-frozen 22-24 days after implantation. Following homogenization and clearing by centrifugation, 1.5 mg of lysate per sample was immunoprecipitated overnight at 4°C with 0.1 mg of anti-H2-K^b (clone Y3, BioXCell) and 0.1 mg of anti-H2-D^b (clone 28-14-8S; hybridoma from ATCC) bound to 20 μL FastFlow Protein A sepharose beads (GE Healthcare). Beads were washed with TBS and water and then peptide-bound MHCs were eluted with 10% acetic acid. Peptides were separated from antibody and MHC via 10K molecular weight cut-off filters (PALL life sciences), lyophilized, and stored in -80°C before labeling. For multiplexing, lyophilized peptide-bound MHCs were resuspended in 33 μL of labeling buffer (50% ethanol, 150 mM TEAB) and mixed with 40 μg of pre-aliquoted TMTpro 16plex Label Reagent (Thermo Fisher Scientific) resuspended in 10 μL of anhydrous acetonitrile. Labeling reaction occurred on a shaker for 4.5 hours at room temperature and quenched with 0.3% hydroxylamine. Samples were pooled and dried in SpeedVac centrifuge prior to cleaning up with SP3 protocol as previously described (25 (link)).