LC-MS/MS Analysis: Enhanced English Description for Scientific Research

LC-MS/MS analysis was conducted on a Q Exactive mass spectrometer (Thermo Scientific) coupled with an Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific) high-performance liquid chromatography (HPLC) system. Initially, labeled peptides dissolved in buffer A (0.1% formic acid) were loaded onto a reversed-phase capture column (Thermo Scientific Acclaim PepMap100, 100 µm × 2 cm, nanoViper C18). Subsequently, the labeled peptides were separated using IntelliFlow technology at a flow rate of 300 nl/min in buffer B (84% acetonitrile and 0.1% formic acid), with a run time of 60/90 minutes. The mass spectrometer operated in positive ionization mode, scanning for parent ions within a mass-to-charge ratio (m/z) range of 300-1800. Following each full scan, 20 fragmentation spectra (MS2 scan) were acquired. The MS2 activation method was high-energy collision dissociation (HCD), with an isolation window of 2 m/z. The first and second mass spectrometry resolutions were 70,000 and 17,500 at 200 m/z, respectively. The normalized collision energy was 30 eV, and the underfill was set to 0.1%.