Thermo acclaim pepmap rslc c18 column

Peptides were dissolved in 100% H₂O plus 0.1% Formic acid (FA) then centrifuged at 20,000 x g. Supernatants were tested with LC-MS on the Ultimate RSLC nano 3000 coupled with the Thermo Scientific Q Exactive HFX (Thermo Fisher Scientific). Enriched peptides were separated using the Thermo Acclaim PepMap RSLC C18 column (75 um × 500 mm, 2 μm, Thermo Fisher Scientific) at 40°C. The mobile phase was composed of 100% H₂O plus 0.1% FA (A) and 80% acetonitrile (ACN) plus 0.1% FA (B). The gradient program was set as follows: 0–6 min, 2–10% solution B; 6–51 min, 10–20% solution B; 51–58 min, 20–80% solution B; 58–62 min, 80% solution B; 62–63 min, 80–2% solution B; 63–70 min, 2% solution B. The flow rate was 250 μL/min.
The Orbitrap was applied to detect whole peptides and ion fragments at resolutions of 60,000 and 30,000, respectively. The electrospray voltage was set to 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the iontrap. The m/z range was from 350–1800 for MS scans. The MS fixed first mass was set at 110 m/z. The affinity enrichment and LC-MS/MS analyses were conducted by the Micron Biotechnology Company.