Eksigent microlc system

The peptide samples were dissolved in 2% acetonitrile/0.1% formic acid and analysed using a TripleTOF 5600plus mass spectrometer coupled with the Eksigent microLC system (AB Sciex). Peptides were loaded onto a C18 trap column (5 μm, 100 μm × 20 mm) and eluted at 5 μL min⁻¹ onto a C18 analytical column (3 μm, 300 μm × 150 mm) over a 60 min gradient. The 60 min solvent gradient was 5% B, 0 min; 5%–27% B, 45 min; 27%–50% B, 3 min; 50%–80% B, 2 min; 80% B, 6 min; 80%–5%, 0.5 min; 5%, 3.5 min. The two mobile phases were buffer A (2% acetonitrile/0.1% formic acid/98% water) and buffer B (98% acetonitrile/0.1% formic acid/2% water). For information‐dependent acquisition (IDA), survey scans were acquired in 250 ms and 35 product ion scans were collected at 50 ms per scan. MS1 spectra were collected in the range 350–1500 m/z, and MS2 spectra were collected in the range 100–1500 m/z. Precursor ions were excluded from reselection for 10 s. In the IDA advanced tab, the option “Adjust CE when using iTRAQ reagent” was selected for iTRAQ samples. The resulting MS/MS data were processed using the Proteinpilot v. 4.5 (AB Sciex) search engine with default parameters. Functional annotation of the significantly different proteins was completed by accessing the GO and KEGG databases (Ashburner et al., 2000 (link); Kanehisa et al., 2016 (link)).