Orbitrap analyser

Tryptic peptides were subjected to tandem mass spectrometry with a Q-Exactive HF tandem mass spectrometer (Thermo Fisher Scientific, Les Ulis, France) incorporating an ultrahigh field Orbitrap analyser coupled in line to an Ultimate 3000 chromatography system (Thermo Fisher Scientific).
The tandem mass spectrometer was operated in the data-dependent acquisition mode as previously described (Klein et al., 2016) (link). Briefly, each sample (4 µL) was injected and desalted on a reversephase capillary C18 PepMap TM 100 precolumn (LC-Packing) and then peptides were resolved based on their hydrophobicity on a nanoscale 500-mm C18 PepMap 100 column (5 mm x 300 µm i.d., Thermo Fisher Scientific) using a 90-min gradient from 2.5% to 40% of 80% CH3CN, 19.9% H2O, and 0.1% formic acid. Once separated, mass spectra of the peptides were acquired in the fullscan range of 350-1800 m/z at a resolution of 60,000 and an AGC target of 3.10 6 . Peptide ions with charges of 2+ or 3+ were selected for fragmentation according to a Top20 method consisting of the sequential analysis of each of the 20 most abundant peptide ions. MS/MS mass spectra were acquired with an AGC target set at 10 4 , a loop count of 60 ms, an isolation window of 1.6 m/z, a resolution of 15,000, a dynamic exclusion of 20 s, and a scan performed over a dynamic mass range from the first mass detected up to 15 times the first mass.