LC-MS/MS Analysis: A Detailed Explanation of the Procedure

LC-MS/MS analysis was conducted using a Q Exactive mass spectrometer (Thermo Scientific), coupled to an Easy nLC liquid chromatography system (Proxeon Biosystems, now Thermo Fisher Scientific). The initial step involved loading the labeled peptides onto a reverse-phase capture column (Thermo Scientific Acclaim PepMap100, 100 um*2 cm, nanoViper C18), utilizing Buffer A (0.1% formic acid). Subsequently, the labeled peptides were separated using Buffer B (84% acetonitrile and 0.1% formic acid) and the IntelliFlow technology, maintaining an optimized flow rate of 300 nl/min over a duration of 60/90 minutes. The mass spectrometer operated in positive ion mode, scanning precursor ions within the mass range of 300-1800 m/z. Following each full scan, twenty fragment spectra (MS2 scan) were collected, employing HCD as the MS2 activation type. The isolation window was set at 2 m/z, while the first and second mass spectrometry resolutions were 70,000 and 17,500 at 200 m/z, respectively. A normalized collision energy of 30eV was applied, and the underfill was maintained at 0.1%.