LC-MS/MS analysis

Purified digested peptide fractions were analyzed by nano LC-MS/MS using EASY-nLC 1200 coupled to Q Exactive™ mass spectrometer (Thermo Fisher Scientific). The peptide samples were separated with a 120 min linear gradient from 0% to 55% buffer B (84% acetonitrile, 0.1% Formic acid) on a reverse phase peptide column (Acclaim™ PepMap™ 100 C18, Thermo Fisher Scientific connected) to the EASY capillary C18 column (75μm inner diameter, 10 cm long, three μm resin; Thermo Fisher Scientific) at a flow rate of 300 nl/min. The data-dependent top10 method was used to acquire MS data by choosing the most abundant precursor ions for HCD fragmentation. The full MS survey scans were acquired at a resolution of 70,000, with automatic gain control target 3e6 and a range of m/z 300–1800. HCD spectra scan with a resolution of 17,500 at m/z 200 and the isolation window was two m/z. The dynamic exclusion was 40 s.