Es803 easy spray pepmap c18 column

Peptide and phosphopeptide samples were analyzed using a QExactive Orbitrap mass spectrometer (Thermo Scientific). Dissolved samples were injected using an Easy-nLC 1000 system (Thermo Scientific) and separated on 50 cm ES803 Easy-Spray PepMap C18 Column (Thermo Scientific). The column was equilibrated with 100% solvent A (0.1% formic acid (FA) in water). Peptides were eluted using the following gradient of solvent B (0.1% FA in ACN): 5% to 22% B, 0 -110 min; 22% -35% B, 110 -120 min; 35 -95% B, 120 -125 min at a flow rate of 0.3 µl/min at 50 o C. High accuracy mass spectra were acquired in data-dependent acquisition mode. All precursor signals were recorded in a mass range of 300 -1700 m/z and a resolution of 70000 at 200 m/z. The maximum accumulation time for a target value of 3e6 was set to 120 ms. Up to 12 data dependent MS/MS were recorded using quadrupole isolation with a window of 2 Da and HCD fragmentation with 30% fragmentation energy. A target value of 1e6 was set for MS/MS using a maximum injection time of 250 ms and a resolution of 70000 at 200 m/z. Precursor signals were selected for fragmentation with charge states from +2 to +7 and a signal intensity of at least 1e5. All precursor signals selected for MS/MS were dynamically excluded for 30 s.