The labeled peptide samples were purified and separated by AKTA purification system. The operation methods and solution preparation were performed essentially as described previously [19 (link)]. The whole elution process was monitored at 214 nm and collected every minute. Thirty distillates were collected and neutralized in 10 pools and desalinated in a C18 cartridge. After each fraction was vacuum centrifuged, the sample was dissolved in 40 μL 0.1% trifluoroacetic acid and kept frozen at − 80 °C for mass spectrometry analysis. Each sample was separated by capillary high-performance liquid chromatography (Thermo scientific EASY column (2 cm, 100 μm 5 μm, C18). The chromatography conditions were as follow: Water with 0.1% formic acid (A) and Acetonitirile with 0.1% formic acid (B) as mobile phase. The flow rate was 300 nL per minute and the mobile phase gradient program was used: 0–33 min, from 0 to 40%(B); 33–34 min, from 40 to 100%(B); 34–35 min maintained 100% and then back to 40%. Then, proteins were analyzed by using a Q-Exactive mass spectrometry (Thermo Finnigan) at positive ion mode (parameters: mass range: 300–1800 m/z; Dynamic exclusion: 40.0 s, MS2 Activation Type: HCD, Normalized collision energy: 30 eV).
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