Linear ion trap orbitrap hybrid mass spectrometer

Peptide separation was performed on a surveyor liquid chromatography system (Thermo Finnigan, San Jose, CA, USA), consisting of a degasser, MS pump, and autosampler and equipped with a C18 trap column (RP, 320 μm×20mm, Column Technology) and an analytical C18 column (RP, 75 μm×150mm, Column Technology). After sample loading, the column was washed for 30min with 98% mobile phase A (0.1% formic acid in water) to flush off remaining salt. Peptides were eluted using a linear gradient of increasing mobile phase B (0.1% formic acid in ACN) from 2 to 35% in 120min. A linear ion trap/Orbitrap hybrid mass spectrometer (Thermo Fisher, San Jose, CA, USA) equipped with a NSI nanospray source was used for the MS/MS experiment. Spray voltage applying to the Nano needle was 1.85kV and ion transfer capillary temperature was 160 °C. Normalized collision energy for collision-induced dissociation was 35%. The number of ions stored in the ion trap was regulated by the automatic gain control. The instrument method consisted of one full MS scan from 400 to 2000 m/z followed by data-dependent MS/MS scan of the 10 most-intense ions from the MS spectrum with the following dynamic exclusion settings: repeat count of 2, repeat duration 30 s, exclusion duration 1.5min. The resolution of the Orbitrap mass analyser was set at 100 000 (m/Δm 50% at m/z 400) for the precursor ion scans.