2.8 DDA Spectral Library and DIA analysis by nano-LC-MS/MS
Data-Dependent Acquisition fractions and DIA samples analysis were both performed on a Q Exactive HF X mass spectrometer (Thermo Fisher Scientific) coupled with an Ultimate 3000 RSLCnano system (Thermo Fisher Scientific). A nano-LC column (150 μm × 30 cm, 1.8 μm, 100 Å) was packed in-house for peptide separation at a flow rate of 500 nl/min. For DDA analysis, peptides were loaded to a C18 trap colullmn (300 μm × 5 mm, 5 μm, Thermo Scientific) with buffer A (2% ACN, 0.1% FA) for 5 min, then eluted with a gradient from 5% to 25% buffer B (98% ACN, 0.1% FA) for 155 min, 25% to 30% buffer B for 10 min, and 30% to 80% buffer B for 5 min. The mass spectrometry parameters were set as below: MS scan range 350−1500 m/z; loop count 30; NCE 28; MS resolution 120 000, maximal injection time (MIT) 50 ms; MS/MS HCD scans with resolution 30 000, MIT 100 ms; dynamic exclusion duration 30 s; isolation window 2.0 m/z; intensity threshold 2.0 e4; charge exclusion, exclude 1, 7, 8, >8. For DIA analysis, the same nano-LC system and gradient was used as DDA analysis. The DIA MS parameters were set as below: full scan range 400-1250 m/z at resolution 120000 with MIT 50 ms; DIA isolation window was set to 17 m/z with loop count 50 and automatic MIT, scanned at resolution 30000; stepped NCE: 22.5, 25, 27.5; AGC target 1e6.