Dionex ultimate 3000 rplc nanosystem

Each biological replicate was analyzed as a single-shot sample. Peptides were dissolved in 0.1% formic acid and analyzed on Orbitrap Fusion Lumos LC–MS (Thermo) coupled to a Dionex Ultimate 3000 RPLC nanosystem (Thermo), with Thermo Xcalibur (v4.1.50) software. Samples were loaded onto a loading column (100 µm × 2 cm) and a C18 separating capillary column (75 µm × 15 cm) packed in-house with Luna 3-μm C18(2) bulk packing material (Phenomenex, USA). The HPLC solvent A was 0.1% formic acid in H₂O, and the solvent B was 0.1% formic acid in 80% MeCN. The samples were run using a 120-min gradient method (0 min 2% B; 8 min 2% B; 9 min 10% B; 123 min 44% B; 128 min 99% B; 138 min 99% B; and 139 min 2% B) with a flow rate of 300 nL/min. The Fusion Lumos was operated in data-dependent mode with a cycle time of 3 s. The MS1 was performed with a full-scan m/z range from 350.00 to 1,600.00 and a mass resolution of 60,000. The maximum injection time was set to 50 ms with an AGC target of 4e5. The MS/MS fragmentation was performed using a quadruple isolation window of 1.6 m/z and the HCD collision mode (30% energy), with the orbitrap resolution set to 15,000, maximum injection time set to 30 ms, and AGC target set to 5e4.