Tmtpro 16plex label reagent

After 56 °C sterilization for 30 min (29 (link)), 5 μl of individual or pooled serum sample was denatured in 50 μl buffer containing 8 M urea in 50 mM Tris-Cl (pH 7.4) at 32 °C for 30 min. Then the sample was reduced with 10 mM dithiothreitol at 37 °C for 1 h, followed by alkylation for 30 min with 40 mM iodoacetamide in darkness at room temperature. After 4 times dilutions with 50 mM NH₄HCO₃, trypsin (Promega, 1/50 of protein amount) was added to the sample twice for digestion at 37 °C for 12 h and additional 4 h, respectively. The peptides were acidified with 0.1% trifluoroacetic acid, desalted with a homemade C18 (Agela Technologies, Tianjin, China) column, and dried by speed-vac. For each sample, the peptides were resolved with 200 μl of 200 mM triethylammonium bicarbonate (TEAB), and 25 μg of peptide was labeled with TMTpro 16plex label reagents (Thermo Fisher Scientific, San Jose, USA) according to the recommendations of the manufacturer. The sequentially collected samples from the same patient were arranged into the same TMT, and samples from different groups were included in each TMT (as shown in Table S2, with the pooled sample as the internal standard). The samples from each TMT were combined and dried by speed-vac.

The proteome analysis was performed similar as previously described (Shen et al., 2020 (link)). Digested peptides were cleaned-up and labeled using TMTpro 16plex label reagents (Thermo Fisher Scientific, San Jose, USA). Peptides were separated into 30 fractions, which were later combined into 15 fractions. Subsequently, the fractions were dried, redissolved in 2% ACN/0.1% formic acid. All the samples were analyzed using liquid chromatography (LC)-coupled tandem mass spectrometry (MS/MS) with a data-dependent acquisition mode on an Orbitrap 480 (Thermo Fisher Scientific, San Jose, USA). During each acquisition, peptides were analyzed using a 30 min-long LC gradient (from 7 to 30% buffer B). The m/z range of MS1 was 375–1,800, with a resolution of 60,000, normalized Automatic Gain Control (AGC) target of 300%, maximum ion injection time (max IT) of 50 ms, and compensation voltages of −48 V and −68 V for FAIMS Pro™. MS/MS experiments were performed with a resolution of 30,000, normalized AGC target of 200%, and 86 ms max IT for Serum and 100 ms for PBMC. The turbo-TMT and the advanced peak determination were enabled.

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)).