Peptide Separation and Characterization

Each labeled peptide was dissolved with 2% acetonitrile (ACN) containing 0.1% formic acid (FA). The online chromatography separation was performed on an Eksigent nanoLC 415 system (SCIEX, USA) using ChromXP C₁₈ column (3 μm, 75 μm × 15 cm, 120 A, ChromXP, Eksigent). The flow rate was 300 nL/min, and the linear gradient was 90 min (Mobile phase A: 2% ACN/0.1% FA, Mobile B: 95% ACN/0.1% FA). A Triple TOF 6600 tandem mass spectrometer (SCIEX, USA) was applied in MS analysis of the separated fractions. According to Zhu et al. [37 (link)], the data were acquired with a 2.4 kV ion spray voltage, 35 psi curtain gas, 12 psi nebulizer gas, and an interface heater temperature of 150 °C. The MS was scanned in IDA (Information-dependent acquisition) mode with a mass range 400-1,500 and an accumulation time of 250 ms. In each IDA cycle, 40 MS/MS spectra (80 ms, mass range 100-1,500) exceeding a threshold of 260 cps with a charge state of 2-4 were acquired. A rolling collision energy setting was applied to all precursor ions for collision-induced dissociation (CID), and the dynamic exclusion time was set for 16 s.