Liquid chromatography–tandem mass spectrometry analysis and data processing

An Easy nLC 1200 ultra-high-performance liquid chromatography coupled to a QExactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) was used for the proteomic analysis with the following settings. Peptides were fractionated using in-house–made 50-cm columns packed with 1.7-µm C18 beads using a binary buffer system, consisting of Buffer A (0.1% formic acid) and Buffer B (80% acetonitrile in 0.1% formic acid). All samples were analyzed over a 90-min gradient, raising the content of Buffer B from 4% to 23% over 65 min, then from 23% to 55% over 13 min, followed by washing with 95% Buffer B. Spectra for full MS were acquired at a resolution of 60,000 at 200 m/z, and the automated gain control target was set to 3 × 10⁶ with a maximum injection time of 20 ms. The dynamic exclusion was set to 20 s. The MS2 measurements were acquired using a resolution of 15,000 at 200 m/z with a top 22 data-dependent mode. Here the automated gain control target was set to 1 × 10⁵ with an injection time of 22 ms. The normalized collision energy in the higher-energy collisional dissociation cell was 25.

The raw data were analyzed using the Andromeda search engine implemented in the MaxQuant software 1.5.3.8 (Cox et al., 2011). Parameters in MaxQuant were set to default with trypsin selected as protease for digestion. A mouse database from Uniprot (16.06.17) with contaminants was used for peptide and protein identification.