LC-MS/SM analysis of digested peptides was performed on an Orbitrap Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen) coupled to an EASY-nLC 1200 (Thermo Fisher Scientific). Peptides were loaded and separated at 250 nl/min on a home-made C18 50 cm capillary column picotip silica emitter tip (75 μm diameter filled with 1.9 μm Reprosil-Pur Basic C18-HD resin, (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany)) equilibrated in solvent A (2% ACN, 0.1% FA). Peptides were eluted using a gradient of solvent B (80% ACN, 0.1% FA) from 3% to 6% in 5 min, 6% to 29% in 130 min, 29% to 56% in 26 min, 56% to 90% in 5 min (total length of the chromatographic run was 180 min including high ACN level steps and column regeneration). Mass spectra were acquired in data-independent acquisition mode with the XCalibur 4.1.31.9 software (Thermo Fisher Scientific, Bremen).
Each cycle was built up as follows: one full MS scan at resolution 30 000 (scan range between 400 and 1200 m/z), AGC was set at 3*106, ion trap was set at 50 ms. All MS1 was followed by 40 isolation windows of 20 m/z, covering the MS1 range from 400 m/z to 1200 m/z. The AGC target was 2*105, and NCE was set to 27. All acquisitions were done in positive and profile mode.
Each cycle was built up as follows: one full MS scan at resolution 30 000 (scan range between 400 and 1200 m/z), AGC was set at 3*106, ion trap was set at 50 ms. All MS1 was followed by 40 isolation windows of 20 m/z, covering the MS1 range from 400 m/z to 1200 m/z. The AGC target was 2*105, and NCE was set to 27. All acquisitions were done in positive and profile mode.