LC-MS/MS-based unlabeled quantitative proteomics

The desalted peptide solution was freeze-dried and dissolved in 0.1% formic acid for injection into a custom-made reverse-phase analytical column (15 cm long, 75 μm inner diameter). Peptides were separated using a linear gradient of mobile phase buffer B (0.1% formic acid in 98% acetonitrile) on an EASY-nLC 1000 UPLC system (Thermo Fisher Scientific). The gradient began at 6% B, increased to 23% B over 26 min, then to 35% B over 8 min, climbed to 80% B over 3 min, and was maintained at 80% B for 3 min. Eluted peptides were loaded into a nano-electrospray ionization (NSI) source and analyzed using tandem mass spectrometry on a Q Exactive™ Plus (Thermo Fisher Scientific) coupled online with the UPLC. The applied spray voltage was 2.0 kV. Full scan MS detected peptides in the m/z range of 350 to 1800 at a resolution of 70,000 in the Orbitrap. Subsequently, selected peptides were detected at a resolution of 17,500 with a normalized collision energy (NCE) of 28. MS analysis was performed in data-dependent scan mode alternating between one MS scan and 20 MS/MS scans, with a dynamic exclusion set to 15.0 s. Automatic gain control (AGC) target and fixed first mass were set to 5E4 and 100 m/z, respectively.