Easy nlc 1200 high

LC-MS/MS data acquisition was performed on a Fusion Lumos mass spectrometer (Thermo Scientific, Germany) coupled with an EASY-nLC 1200 high-performance liquid chromatography system (Thermo Scientific, Germany). For DDA-MS and DIA-MS modes, the same LC settings were used for retention time stability. The digested peptides were dissolved in 0.1% formic acid and loaded onto a trap column (75 µm × 2 cm, 3 µm, C18, 100 A˚), and the eluent was transferred to a reversed-phase analysis column (50 µm × 250 mm, 2 µm, C18, 100 A˚) with an elution gradient of 5-30% phase B (79.9% acetonitrile, 0.1% formic acid, flow rate of 0.3 μL/min) for 90 min. To enable fully automated and sensitive signal processing, the calibration kit (iRT kit, Biognosys, Switzerland) was spiked at a concentration of 1:20 v/v in all samples。 For the generation of the spectral library, 10 peptide fractions were analyzed in DDA-MS mode.
The parameters were set as follows. The full scan was acquired from 350 to 1 550 m/z at 60 000, and the cycle time was set to 3 s (top speed mode). The auto gain control (AGC) was set to 1e6, and the maximum injection time was set to 50 ms. The MS/MS scans were acquired in the Orbitrap at a resolution of 15000 with an isolation window of 2 Da and collision energy at 32% (HCD). The AGC target was set to 5e4, and the maximum injection time was 30 ms.