2.4 Mass spectrometry analysis
The fraction was then centrifuged at 12000 g for 20 min at 4 °C after being lyophilized and redissolved with 0.1% formic acid in 2% acetonitrile (solvent A). A C18 (150 μm × 15 cm, Evosep) was used in conjunction with the Orbitrap Exploris 480 (Thermo, USA) and Evosep One (Evosep). Data were acquired by Thermo Xcalibur 4.0 (Thermo, USA). The peptide mixture underwent gradient elution using mobile phases of 0.1% formic acid (A) and 100% acetonitrile 0.1% formic acid (B) at a flow rate of 300 nL·min–1. The specific gradients are as follows: 0–2 min, 5–5% solvent B; 2–30 min, 5–38% solvent B; 30–40 min, 38–90% solvent B; 40–44 min, 90–100% solvent B. The mass spectrum parameters were set as follows: scanning range, 350–1500m/z for full scan; acquisition mode, data dependent acquisition (DDA); MS1 resolution, 60000; maximum injection time, 25 ms; fragmentation mode, high energy collisional dissociation (HCD); MS/MS resolution, 15000; maximum injection time, 22 ms; fixed first mass, 110m/z ; minimum automatic gain control (AGC), 8 e3; intensity threshold, 8.3 e4; dynamic exclusion time, 30 s.