High-resolution LC-MS/MS analysis of peptides

An aliquot of 10 µL of the sample was used for the LC-MS/MS analysis as previously described [22 (link)]. In short, an online installed C18 trapping column was used for desalting. This was performed on a Thermo Scientific (Dionex, Sunnyvale, CA, USA) Ultimate 3000 Rapid Separation UHPLC system. and a PepSep C18 analytical column (15 cm, ID 75 µm, 1.9 µm Reprosil, 120 Å) was used to separate the peptides with a flowrate of 300 nL/min, solvent A (100% H₂O, 0.1% FA) and solvent B (80% ACN, 0.1% FA) and a 110 min linear gradient from 5% to 32% ACN with 0.1% FA as follows: 0–3 min 0% B; 3 min 5% B; 103 min 27.5% B; 113 min 40% B; 114–120 min 95% B; 121–140 min 3% B. The UHPLC system was coupled to a Q ExactiveTM HF mass spectrometer (Thermo Scientific) with Nanospray Flex source. Mass spectra were acquired in positive ionization mode, with a full MS scan between 250–1250 m/z at resolution of 120,000 followed by MS/MS scans of the top 15 most intense ions at a resolution of 15,000 in DDA mode.