Zorbax 300sb c18 reverse phase column

Peptides were separated by reverse‐phase nanoflow high‐performance liquid chromatography (nano‐HPLC) and quantitated on an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). Data‐dependent analysis was performed at a resolution of 60,000 and with the top 20 most intense ions selected from each MS full scan, with dynamic exclusion set to 90 s if m/z acquisition was repeated within a 45‐s interval. In each scan cycle, the top 20 fragmentation spectra were acquired in the collision‐induced dissociation mode. For peptide separation, an Agilent ZORBAX 300SB‐C18 reverse‐phase column (150 mm × 75 μm inner diameter) and a 240‐min gradient of 2–90% acetonitrile and 0.1% formic acid were used. The experiment was conducted twice (biological replicates) and four technical replicates for each sample were collected (repeat mass spectrometry measurements). The data for technical replicates were combined during the computational analysis, while the biological replicates were kept separately.