2.5. LC-MS/MS Analysis

LC-MS/MS analysis was carried out on an Ultimate 3000 RSLC coupled with a Q Exactive HF-X (both Thermo Scientific, Waltham, MA, USA). Aliquots of 1.5 µg peptides were injected onto a trap column (PepMap 100 C18, 100 μm × 2 cm, 5 μM particles, Thermo Scientific, Waltham, MA, USA) at a flow rate of 5 μL/min mobile phase A (0.1% formic acid and 1% acetonitrile in water). Peptide separation was performed with an EASY-Spray column (PepMap RSLC C18, 75 μm × 50 cm, 2 μm particles, Thermo Scientific, Waltham, MA, USA) and a flow rate of 250 nL/min. A two-step gradient was used: first from 3% mobile phase B (0.1% formic acid in acetonitrile) to 25% B in 160 min and second a 10 min ramp to 40% B (A: 0.1% formic acid in water). Peptides were analyzed in the data-dependent acquisition mode with up to 15 MS/MS scans per cycle.