Anti h2 kb clone y3

Peptide MHC isolation was performed as described previously^{33 (link)}. Healthy lung tissue or tumor bearing lung tissue was homogenized and cleared as described for Streptactin purification. Per sample, 1 mg of Anti-H2-K^b (clone Y3, BioXCell) was bound to 20 μL (bed volume) FastFlow Protein A Sepharose beads (GE Healthcare) by incubating for 1 hour at 4°C. Beads were then washed with lysis buffer and samples were incubated for 2-4 hours rotating at 4°C. Beads were then centrifuged at 1,000 rpm, washed twice with MEB, twice with 1× TBS, and eluted with 10% acetic acid at room temperature. Eluate was then filtered using 10 kDa MWCO spin filters (PALL Life Science), which were passivated with 0.1% BSA and acidified with 10% acetic acid prior to filtration. Filtered peptides were then further purified with 8 μg binding capacity C18 tips (Pierce) prior to LC/MS-MS analysis. . For multiplexing, lyophilized pMHC were resuspended in 33 μl of labeling buffer (50% ethanol, 150 mM TEAB) and mixed with 40 μg of pre-aliquoted TMT 6plex (Thermo Scientific) resuspended in 10ul of anhydrous acetonitrile. Labeling reaction was carried out on a shaker for 1 hour at room temperature and quenched with 0.3% hydroxylamine. Samples were combined and dried in SpeedVac prior to being cleaned up with SP3 protocol as previously described (Stopfer et. al. 2020).