Xbridge peptide beh c18 column

The peptides of 100 μg taken from each sample were labelled using iTRAQ reagents (AB SCIEX, USA) following the manufacturer's protocol. The labelled peptides were mixed, and then, chromatography was conducted with Agilent 1260 Infinity II Liquid Chromatography System (Agilent, USA). Labelled peptide mixtures were reconstituted within high pH RP buffer A (10 mΜ HCOONH₄, 5% acetonitrile, pH 10.0) and loaded by a manual injector onto a 4.6 mm × 100 mm XBridge Peptide BEH C18 column (Thermo Scientific, USA). The peptides were eluted at a flow rate of 1 mL/min. The gradient was 0% high pH RP buffer B (10 mΜ HCOONH4, 85% acetonitrile, pH 10.0) for 5 minutes, 0%‐7% buffer B for 25 minutes, 7%‐40% buffer B for 30 minutes, 40%‐100% buffer B for 65 minutes and 100% buffer B for 70 minutes. The absorbance value at 214 nm was detected, and fractions were collected at intervals of one minute, amounting to 36 fractions. Those fractions were pooled for each sample and lyophilized by a vacuum centrifuge. Fractions for each sample were then resuspended in 0.1% formic acid (FA) (Buffer A of the mobile phrase for LC‐MS/MS).

The peptides at 100 μg extracted from each sample were labelled using iTRAQ reagents (AB SCIEX, USA) following the manufacturer’s protocol. The labelled peptides were mixed, and then, chromatography was conducted with Agilent 1260 Infinity II Liquid Chromatography System (Agilent, USA). Labelled peptide mixtures were reconstituted within high-pH RP buffer A (10 mΜ HCOONH₄, 5% acetonitrile, pH 10.0) and loaded by a manual injector onto a 4.6 mm × 100 mm X Bridge Peptide BEH C18 column (Thermo Scientific, USA). The peptides were eluted at a flow rate of 1 ml/min. The gradient condition was 0% high-pH RP buffer B (10 mΜ HCOONH4, 85% acetonitrile, pH 10.0) for 5 min, 0–7% buffer B for 25 min, 7–40% buffer B for 30 min, 40–100% buffer B for 65 min, and 100% buffer B for 70 min. The absorbance value at 214 nm was detected, and fractions were collected at intervals of 1 min, amounting to 36 fractions. Those fractions were pooled for each sample and lyophilised by vacuum centrifugation. Fractions for each sample were then resuspended in 0.1% formic acid (FA) (Buffer A of the mobile phrase for LC-MS/MS).

Proteins were extracted from FaDu and FaDu/DDP cells to obtain a peptide solution by filter-aided sample preparation. The pooled peptides were fractioned by reversed-phase chromatography. The peptide mixture was diluted with buffer A (0.1% formic acid) and loaded onto an XBridge Peptide BEH C18 Column (Thermo Fisher Scientific). The peptides were eluted with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 1 ml/min. LC-MS/MS analysis was performed on Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) that was coupled to Easy nLC (Thermo Fisher Scientific) for 90 min.