The peptides prepared in the previous section were analyzed by the EASY-nLC 1000 UPLC, which was equipped with a reversephase analytical Acclaim PepMap RSLC column (75 μm × 150 mm, 3 μm particles, ThermoFisher) [7 (link), 11 (link)]. The peptides MS/MS analysis was carried out by tandem MS/MS in Quadrupole-Orbitrap mass spectrometer (Q Exactive™, ThermoFisher) coupled online to UPLC system. The resolution of intact peptides in the Orbitrap was 70,000 and the resolution of ion fragments in the Orbitrap was 17,500. The normalized collision energy (NCE) was set at 28. In the MS survey scanning, a data-dependent program was executed that alternated between 1 scan and subsequent 20 scans for the top 20 precursor ions which exceeded ion number of 5E3 with dynamic exclusion of 15.0 s [8 (link), 35 (link)]. The overfilling of the Quadrupole-Orbitrap was realized by automatic gain control (AGC). For MS scanning, the scanning range of mass spectrum was 350 ~ 1800. The fixed first mass was set at 100 m/z [17 (link), 18 (link), 20 (link), 35 (link)]. The electrospray voltage of MS/MS was set at 2.0 kV.
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