Q extractive mass spectrometer

LC-MS analysis was performed using a Q Extractive mass spectrometer coupled to an Easy nLC system (Thermo Fisher Scientific). Peptide from each fraction was loaded onto a C18-reversed phase column (12 ​cm long, 75 ​μm ID, 3 ​μm) in buffer A (2% acetonitrile and 0.1% formic acid) and separated with a linear gradient in buffer B (90% acetonitrile and 0.1% formic acid) at a flow rate of 300 ​nL/min for 90 ​min. The linear gradient was set as follows: 0–2 ​min, 2–5% buffer B; 2–62 ​min, 5–20% buffer B; 62–80 ​min, 20–35% buffer B; 80–83 ​min, 35–90% buffer B; and 83–90 ​min, buffer B maintained at 90%. MS data were acquired using a data-dependent top 15 method by dynamically choosing the most abundant precursor ions from the survey scan (300–1800 m/z) for higher-energy collisional dissociation (HCD) fragmentation. The target value was determined based on predictive automatic gain control (pAGC). The AGC target values of 1e⁶ and a maximum injection time of 50 ​min were used for full MS, while the target AGC value of 1e⁵ and a maximum injection time of 100 ​min were used for MS2. The dynamic exclusion duration was 30 ​s. Survey scans were acquired at a resolution of 70,000 ​at ​m/z 200, and the resolution of the HCD spectra was set to 35,000 ​at ​m/z 200. The normalized collision energy was 30. The instrument was run in the peptide recognition mode enabled.