Peptide Analysis via Q Exactive MS

The enriched peptides were analysed with the EASY‐nLC1000 system (Thermo Finnigan) connected to a Q Exactive mass spectrometer (Thermo Finnigan). Briefly, peptide samples were dissolved in 0.1% formic acid, directly loaded onto a reverse‐phase precolumn (Thermo EASY column SC200, 150 μm × 100 mm). A reverse‐phase analytical column (Thermo EASY column SC001 traps, 150 μm × 20 mm) was used for peptide separation, as described previously (Li, Sun, et al., 2016 (link)). The resulting peptides were evaluated with the Q Exactive mass spectrometer for 120 min. The mass spectrometer was operated in positive ion mode. MS data were acquired using a data‐dependent procedure, dynamically choosing the most abundant precursor ions from the survey scan (350–1800 m/z) for high‐energy collision dissociation fragmentation. Survey scans were acquired at a resolution of 70,000 at m/z 200 and the resolution for high‐energy collision dissociation spectra was set to 17,500 at m/z 200. Automatic gain control was used to prevent overfilling of the ion trap and 5 × 10⁴ ions were accumulated for generating the MS/MS spectra. Each LC–MS/MS analysis was repeated three times to reduce technical variation.