Aqua c18 beads

Phosphopeptides were redissolved in 100 μL 0.1% formic acid and centrifuged at 13,000× g and 4 °C for 15 min to collect their supernatants. The peptides were injected into an Easy C18 column (100 μm I.D. × 20 mm, 5 μm) using an autosampler, and each sample was analyzed in triplicate. The peptides were analyzed using an EASY-nLC 1000 coupled Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The samples were loaded onto a trap column (75 μm inner diameter; fused silica filled with 3.0 μm Aqua C18 beads; Thermo Fisher Scientific) in buffer A (0.1% formic acid) and buffer B (0.1% formic acid in acetonitrile) at a flow rate of 0.35 μL/min. A gradient of 3–90% buffer B was applied for 120 min, followed by a 10-min gradient of 90%. Data-dependent ion signals were collected and run with the following settings: resolution 70,000; automatic gain control target, 3 × 10⁶; maximum injection time, 20 ms; scan range, m/z 300–1800. The resolution of the MS/MS was 17,500, and the loop count was 10. Charge exclusion: 1, >8, and dynamic exclusion: 20 s. Thermo Fisher Scientific Xcalibur software version 2.2 was used for processing raw data.