μ precolumn c18 pepmap 100

An UltiMate 3000 RSLC nano LC system (Dionex) fitted with a trapping cartridge (μ-Precolumn C18 PepMap 100, 5 μm, 300 μm i.d. × 5 mm, 100 Å) and an analytical column (nanoEase M/Z HSS T3 column 75 μm × 250 mm C18, 1.8 μm, 100 Å, Waters) was used, coupled directly to an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo). Peptide samples were loaded onto the precolumn with a constant flow rate of 30 μl/min for 6 min using 0.05% v/v trifluoroacetic acid in water. Subsequently, peptides were eluted via the analytical column (Solvent A: 0.1% formic acid in water) with a constant flow of 0.3 μl/min, with increasing percentage of solvent B (0.1% formic acid in acetonitrile). The peptides were introduced into the Fusion Lumos via a Pico-Tip Emitter 360 μm OD × 20 μm ID; 10 μm tip (New Objective) and an applied spray voltage of 2.4 kV. The capillary temperature was set to 275 °C. Full mass scan (MS1) was acquired with a mass range of 375 to 2000 m/z in profile mode in the orbitrap with a resolution of 120,000. The filling time was set at a maximum of 50 ms. Data-dependent acquisition was performed with the resolution of the Orbitrap set to 30,000, with a fill time of 86 ms. A normalized collision energy of 34 was applied. MS2 data were acquired in profile mode.