Proteomic Analysis of CDN-treated 116 cells

Peptides were resuspended in 0.1% formic acid and analyzed with Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific，USA) equipped with an Easy-nLC 1200 high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific, USA). Peptides were subsequently loaded onto a homemade trap column (3 μm particle size, 120 Å pore size, 100 μm × 2.0 cm, SunChrom, USA) and separated on a homemade analytical microcolumn (1.9 μm particle size, 120 Å pore size, 150 μm × 15.0 cm, SunChrom, USA). The separation employed a 60-min linear gradient of mobile phase B from 7% to 40% mobile phase B (0.1% formic acid in acetonitrile) at a constant flow rate of 600 nL/min. The gradient profile was as follows: 7–10% B for 3 min, 10–25% B for 39 min, 25–40% B for 11 min, 40–95% B for 1 min, and 95% B held for 6 min. Mass spectrometry analysis was performed in a data-dependent acquisition (DDA) mode. Full MS scans were acquired at a resolution of 120,000 with an automatic gain control (AGC) target of 5e5. A top-speed mode was used with a 3-second cycle time. The most intense precursor ions were isolated by the quadrupole with a 1.6Th window. Higher-energy collision dissociation (HCD) was performed with a normalized collision energy (NCE) of 32%. The fragment ions were detected at a resolution of 15,000 with an MS2 AGC target of 5e4. A dynamic exclusion was set to 30 seconds.