Proteomics Workflow with LTQ-Orbitrap

Each biological replicates was analyzed by duplicate resulting 24NanoLC-MS/MS experiments (experimental replicates). The 24 resulting peptide mixtures were performed using an LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher) coupled to an UltiMate 3000 LC system (Dionex-LC Packings) in conditions similar to those previously described [48 (link),49 (link)]. Peptide mixtures (10 μl) were loaded and desalted on-line in a reverse phase precolumn (Acclaim PepMap 100 C18, 5μm bead size, 100 Å pore size, 5mm x 300μm) from LC Packings at a flow rate of 0.3 μl/min with a gradient of CH₃CN/0.1% formic acid prior to injection into the ion trap mass spectrometer. Peptides were resolved using a 90 min gradient from 5% to 60% of solvent B (0.1% HCOOH/80% CH₃CN). Solvent A was 0.1% HCOOH/100% H₂O. Full-scan mass spectra were measured from m/z 300 to 1,700 with the LTQ-Orbitrap XL mass spectrometer in data-dependent mode using the TOP7 strategy. In brief, a scan cycle was initiated with a full scan of high mass accuracy in the Orbitrap. This was followed by MS/MS scans in the linear ion trap of the seven most abundant precursor ions, with dynamic exclusion of previously selected ions.