Reprosil pur c18 aq resin

The LC-MS/MS analyses of the HLA and the tryptic peptides were performed with a Q-Exactive-Plus mass spectrometer fitted with either with Easy nLC 1000 capillary HPLC (Thermo-Fisher Scientific) or with Ultimate 3000 RSLC nano-capillary UHPLC (Thermo-Fisher Scientific). The reversed phase chromatographies were performed with home-made 30 cm long, 75 μm inner diameter, packed with 3.5 μm silica ReproSil-Pur C18-AQ resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany), as in Ishihama et al. (87 (link)). The HLA peptides were eluted using a linear gradient of 5–28% of acetonitrile in 0.1% formic acid, at a flow rate of 0.15 μl/min for 2 h. Data was acquired using a data-dependent “top 10” method, fragmenting the peptides by higher-energy collisional dissociation (HCD). Full scan MS spectra were acquired at a resolution of 70,000 at 200 m/z with a target value of 3 × 10⁶ ions. Fragmented masses were accumulated to AGC (automatic gain control) target value of 10⁵ with a maximum injection time of 100 ms. No fragmentation was attempted for HLA peptides with unassigned precursor charge states. The peptide match option was set to Preferred. The normalized collision energy was set to 25% and MS/MS resolution was 17,500 at 200 m/z. Fragmented m/z values were dynamically excluded from further selection for 20 s.

Mass spectrometry analysis was performed on a Q Exactive Plus Mass Spectrometer (Thermo-Fisher Scientific, Waltham, MA, USA) using the positive ion mode and full MS scan repetitively followed by higher-energy collision dissociation (HCD) of the 10 most dominant ions selected from the full MS scans. Reversed-phase chromatography was performed with home-made, about 30 cm long, 75 μm inner diameter capillaries packed with 3.5 μm silica ReproSil-Pur C18-AQ resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany), as previously described [84 (link)]. Peptides were eluted using a linear gradient of 5–28% acetonitrile in 0.1% formic acid at a flow rate of 0.15 μL/min for 3 h. Full-scan MS spectra were acquired at a resolution of 70,000 at 200 m/z with a target value of 3 × 10⁶ ions. Fragmented masses were accumulated to an automatic gain control (AGC) target value of 10⁵ with a maximum injection time of 100 ms. Fragmentation was performed for charge states of 2 to 7. The peptide match option was set to Preferred. The normalized collision energy was set to 25%, and MS/MS resolution was 17,500 at 200 m/z. Fragmented m/z values were dynamically excluded from further selection for 20 s.

We commissioned Beijing Biotech-Pack Co. Ltd. (Beijing, China) to conduct mass spectrometry analysis. Proteins A and B recovered from the SDS-page gel were digested by trypsin and an Ultimate 3000 system (Thermo Fisher Scientific, Waltham, MA, USA) interfacing with a Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer system (Thermo Fisher Scientific, Waltham, MA, USA) was used for the LC-MS/MS analysis. The peptides were reconstituted with 10 μL of 0.1% formic acid (0.1% FA, 2% CAN), and 5 μL of the solution was loaded and separated on a 150 μm × 15 cm in-house-made column packed with a reversed-phase ReproSil-Pur C18-AQ resin (1.9 μm, 100 Å, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). The mobile phase for chromatographic separation consisted of 0.1% formic acid in 2% acetonitrile (A) and 0.1% formic acid in 80% acetonitrile (B). The flow rate was set to 600 nL/min. The gradient was set as follows: 0–5 min, 6–9% B; 5–20 min, 9–14% B; 20–50 min, 14–30% B; 50–58 min, 30–40% B; 58–60 min, 40–95% B. The MS spectra were acquired from 350 to 1800 m/z with a resolution of 70,000 FWHM, and MS/MS scan resolution of 75,000. The top 20 most intense peptide ions from the preview scan in the Orbitrap were selected. The raw MS files were analyzed and searched against a target protein database based on the species of the samples using MaxQuant (1.6.2.10).

Nanoflow UPLC utilized the Ultimate 3000 system (ThermoFisher Scientific, Waltham, MA, USA) and the nanocolumn was a 150 μm×15 cm in-house made column packed with a reversed-phase ReproSil-Pur C18-AQ resin (1.9 μm, 100 Å, Dr. Maisch GmbH, Germany). The parameters were set as follows: loaded sample volume, 5 μL; mobile phase A, 0.1% formic acid in water; mobile phase B, 0.1% formic acid in acetonitrile; total flow rate, 600 nL/min; LC linear gradient, from 4% to 8% B for 2 min, from 8% to 28% B for 43 min, from 28% to 40% B for 10 min, from 40% to 95% B for 1 min, and from 95% to 95% B for 10 min.