Acclaim pepmap 100 c18 rslc column

The samples in 1% (vol/vol) aqueous FA were loaded (2.5 µL) onto a 75 µm i.d. × 50 cm Acclaim® PepMap 100 C18 RSLC column (Thermo-Fisher Scientific) on an EASY nano-LC (Thermo Fisher Scientific). The column was equilibrated using constant pressure (700 bar) with 11 μL of solvent A (1% (vol/vol) aqueous FA). The peptides were eluted using the following gradient programme with a flow rate of 300nL/min and using solvents A and B (1% (vol/vol) FA/MeCN): solvent A containing 5% B for 5 min, increased to 23% B over 105 min, to 35% B over 20 min, to 95% B over 1 min and constant 95% B for 19 min. The data were acquired in data-dependent acquisition (DDA) mode. The MS1 scans were acquired with the Orbitrap™ mass analyser over m/z = 350 to 1500 and resolution set to 70,000. Twelve data-dependent high-energy collisional dissociation spectra (MS2) were acquired from each MS1 scan with a mass resolving power set to 35,000, a range of m/z = 100–2000, an isolation width of 1.2 m/z, and a normalized collision energy setting of 32%. The maximum injection time was 60 ms for parent-ion analysis and 120 ms for product-ion analysis. The ions that were selected for MS2 were dynamically excluded for 40 sec. The automatic gain control (AGC) was set at a target value of 3e6 ions for MS1 scans and 1e5 ions for MS2. Peptide ions with charge states of one or ≥7 were excluded for HCD acquisitions.