Pepmap 100 c18 nanolc column

Peptides were analyzed with an LTQ-Orbitrap XL hybrid mass spectrometer (Thermofisher, Villebon sur Yvette, France) coupled to an ultimate 3000 nanoLC system (Thermo) operated in data-dependent mode essentially as previously described [35 (link)]. The digests (50 µL) were loaded with a specific large injection loop and desalted online on a reverse phase PepMap100 C18 µ-precolumn (5 µm, 100 Å, 300 µm i.d. × 5 mm, Thermofisher, Villebon sur Yvette, France) and resolved on a nano scale PepMap 100 C18 nano LC column (3 µm, 100 Å, 75 µm i.d. × 50 cm, Thermofisher) at a flow rate of 0.3 µL.min⁻¹ with a gradient of CH₃CN, 0.1% formic acid prior to injection into the ion trap mass spectrometer. Peptides were resolved using a 60-min gradient from 2.5% to 50% solvent B (0.1% HCOOH/20% H₂O/80% CH₃CN) against solvent A (0.1% HCOOH/100% H₂O). A top 5 strategy was used for the acquisition of MS/MS essentially consisting in a full MS scan, followed by fragmentation and MS/MS scan on the 5 most abundant ion precursors. Full scan mass spectra were measured from m/z 300 to 1800 in the Orbitrap analyzer at 30,000 resolution. The MS/MS scans were triggered in the linear ion trap at a resolution of 10,000 with a minimum signal required set at 10,000, potential charge states of 2+ and 3+, and with a 10 s dynamic exclusion of previously selected ions.

Peptides were analyzed with a Q-Exactive HF (Thermo Scientific) tandem mass spectrometer coupled to an ultimate 3000 nano-LC system (Thermo Scientific). Peptides were desalted on a reverse-phase PepMap 100 C18 μ-precolumn (5 mm, 100 Å, 300 mm i.d. × 5 mm, Thermo Scientific) and separated on a nanoscale PepMap 100 C18 nanoLC column (3 mm, 100 Å, 75 mm i.d. × 50 cm, Thermo Scientific) at a flow rate of 0.3 μL/min using a 30 min gradient (2.5% B from 0 to 1.5 min, 2.5–25% B from 1.5 to 26.5 min, and 25–40% B from 26.5 to 30 min) of mobile phase A (0.1% HCOOH/100% H₂O) and phase B (0.1% HCOOH/80% CH₃CN). The mass spectrometer operated in data-dependent acquisition mode with a Top20 strategy, i.e., the 20 most abundant precursor ions were serially selected for fragmentation. Full-scan mass spectra were acquired from 350 to 1800 m/z. Only peptides with 2 or 3 positive charges were selected for fragmentation with a dynamic exclusion time of 10 s and an isolation window of 1.6 m/z.