LC-MS/MS analysis

Proteome samples were analyzed with a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an EASY-nLC 1000 system (Thermo Fisher Scientific). Reversed-phase separation was performed with a 3 cm trap column (packed in-house with 200 Å C18 magic bead, 5 μm) (Bruker) and a 65 cm analytical column (100 Å C18 magic bead, 5 μm), using mobile phase of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). A linear separation gradient with 2–10% solvent B for 1 min, 10–30% solvent B for 89 min, and 30–80% solvent B for 1 min was used. The flow rate was set to 300 nl/min. Data-dependent acquisition analysis with Q Exactive Plus included MS¹ analysis with scan range 400–2000 m/z, scan resolution 70 000 and automatic gain control (AGC) target value 1 × 10⁶, and MS/MS analysis for the top 20 most abundant precursor ions detected in MS¹ scans. MS/MS analysis included isolation window 1.6 m/z, fragmentation energy 25 NCE, scan resolution 17 500, AGC target value 5 × 10⁴. Charge exclusion for 1+ and unassigned ions was enabled. Dynamic exclusion of 30 s was used.