The peptides were re-dissolved in solvent A containing 0.1% formic acid and analyzed by on-line nanospray LC-MS/MS on Q Exactive™ (Thermo Fisher Scientific, MA, USA) coupled to EASY-nano-LC 1200 system (Thermo Fisher Scientific, MA, USA). 2.5 μL peptide sample was loaded, and separated with 90 min-gradient, from 5 to 35% B (B: 0.1% formic acid in ACN). The column flow rate was maintained at 300 nL/min. The electrospray voltage of 2 kV versus the inlet of the mass spectrometer was used.

The mass spectrometer was run under data dependent acquisition mode, and automatically switched between MS and MS/MS mode. The parameters was: (1) MS: scan range (m/z)=300–1800; resolution=70,000; AGC target=3e6; maximum injection time=60 ms; include charge states=2–7; dynamic exclusion=20s; (2) HCD-MS/MS: resolution=17,500; isolation window=2.2; AGC target=5e4; maximum injection time=80 ms; collision energy=30.

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