LC-MS/MS analysis

Tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a reversed-phase analytical column (15-cm length, 75 μm i.d.). As described previously (13), the gradient comprised of solvent B at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system. Then, the peptides were subjected to the NSI source, followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC, with the following parameter: 2.0 kV electrospray voltage, 350 to 1800 m/z scan range, and resolution of 70,000 for detection. Selected peptides MS/MS and fragments were detected in the Orbitrap at a resolution of 17,500. Depending on the data, a program of 20 MS/MS scans was carried out alternating after one MS scan with 15.0s dynamic exclusion. Automatic gain control (AGC) was programmed at 5E4. The fixed first mass was set at 100 m/z.