Pepmap c18 reversed phase column

The labeled peptide was diluted with solution A (0.1% formic acid, 2% ACN, 98% water) and preseparated with solution B (0.1% formic acid, 2% water, 98% ACN), and different components were collected at different times. The collected peptides were desalted and purified by C18 column according to the manufacturer’s instructions. Desalted peptide mixtures were loaded onto an Acclaim PepMap C18 reversed-phase column (75 μm × 2 cm, 3 μm) and separated with a reversed-phase C18 column (75 μm × 10 cm, 5 μm) mounted on a Dionex UltiMate 3000 Nano LC system. Peptides were eluted using a gradient of solution B (5%, 5%, 30%, 60%, 80%, 100%) at a flow rate of 300 nL/min combined with a Q Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA). The eluates were directly entered into the Q Exactive MS (Thermo Fisher Scientific, Waltham, MA, USA) in positive ion mode and data-dependent manner with a full MS scan from 350–2000 m/z, full scan resolution at 70,000, and MS/MS scan resolution at 17,500. An MS/MS scan with minimum signal threshold 1 × 10⁵, isolation width at 2 Da was completed. The MS/MS acquisition mode was higher collision energy dissociation (HCD). To optimize the MS/MS acquisition efficiency of HCD, normalized collision energy (NCE) was systemically examined with a stepping of 20%.

Peptides were concentrated on a Pepmap C-18 trap column (300 μm ID × 5 mm) and separated on a Pepmap C18 reversed phase column (Dionex, UK) (3 μm particles, 75 μm ID × 250 mm) using a linear gradient over 42 min from 96% A (0.05% formic acid), 4% B (0.05% formic acid, 80% acetonitrile) to 35% A, 65% B and a flow rate of 300 nl/min.