Proteome Analysis by Q-Exactive Mass Spectrometry

All analyses were performed by a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA) coupled with a Nanospray Flex source (Thermo Fisher Scientific, USA) as described earlier [7 (link)]. Samples were redissolved with 0.1% formic acid and separated by a C18 column (15 cm × 75 µm) on an EASY-nLC^TM 1200 system (Thermo Fisher Scientific, USA). The flow rate was 300 nL/min, and the linear gradient was 90 min. Full MS scans were acquired in the mass range of 300–1,600 m/z with a mass resolution of 70,000, and the automatic gain control (AGC) target value was set at 1e6. The ten most intense peaks in MS were fragmented with higher energy collisional dissociation with positive polarity in the data-dependent mode. MS/MS spectra were obtained with a resolution of 17,500 with an AGC target of 2e5 and a max injection time of 80 ms. Fragmentation was performed with normalized collision energy of 32 and dynamic exclusion of 30 s.