Easy spray rslc c18 column

Peptides were separated by nano liquid chromatography (Thermo Fisher Scientific, Ultimate RSLC 3000 or EASY-nLC) coupled in line with a Q Exactive mass spectrometer equipped with an EASY-Spray source (Thermo Fisher Scientific). Peptides were trapped onto a C18 PepMac 100 pre-column (300 µm i.d. × 5 mm, 100 Å, Thermo Fisher Scientific) using solvent A (0.1% formic acid and HPLC-grade water). The peptides were further separated onto an EASY-Spray RSLC C18 column (75 µm i.d., 50-cm length, Thermo Fisher Scientific) using a 60-min linear gradient (15% to 35% solvent B (0.1% formic acid in acetonitrile)) at a flow rate of 200 nl min⁻¹. The raw data were acquired on the mass spectrometer in data-dependent acquisition mode. Full-scan MS spectra were acquired in the Orbitrap (scan range 350–1,500 m/z, resolution 70,000; AGC target, 3 × 10⁶; maximum injection time, 50 ms). The 10 most intense peaks were selected for HCD fragmentation at 30% of normalized collision energy. HCD spectra were acquired in the Orbitrap at resolution 17,500, AGC target 5 × 10⁴ and maximum injection time 60 ms, with fixed mass at 180 m/z. Charge exclusion was selected for unassigned and 1+ ions. The dynamic exclusion was set to 40 s.

Peptides were separated by nanoliquid chromatography (Thermo Fisher Scientific; UltiMate RSLC 3000) coupled in line to a Q Exactive mass spectrometer equipped with an Easy-Spray source (Thermo Fisher Scientific). Peptides were trapped onto a C₁₈ PepMac100 precolumn (300 μm [inside diameter] by 5 mm, 100 Å; Thermo Fisher Scientific) using solvent A (0.1% [vol/vol] formic acid, high-performance liquid chromatography [HPLC]-grade water). The peptides were further separated on an Easy-Spray RSLC C₁₈ column (75-μm inside diameter, 50-cm length; Thermo Fisher Scientific) using a 60-min linear gradient of 15% to 35% solvent B (0.1% [vol/vol] formic acid in acetonitrile) at a flow rate of 200 nL min⁻¹. The raw data were acquired on the mass spectrometer in a data-dependent acquisition (DDA) mode. Full-scan MS spectra were acquired in the Orbitrap (scan range, 350 to 1,500 m/z; resolution, 70,000; AGC target, 3e⁶; maximum injection time, 50 ms). The 10 most intense peaks were selected for higher-energy collision dissociation (HCD) fragmentation at 30% of normalized collision energy. HCD spectra were acquired in the Orbitrap at a resolution of 17,500, automatic gain control (AGC) target of 5e⁴, and maximum injection time of 60 ms, with fixed mass at 180 m/z. Charge exclusion was selected for unassigned and 1⁺ ions. The dynamic exclusion was set to 40 s.

Peptides were separated using nano liquid chromatography (Thermo Fisher Scientific Ultimate RSLC 3000) coupled with a Q Exactive mass spectrometer equipped with an Easy-Spray source (Thermo Fisher Scientific). After separation, peptides were trapped onto a C18 PepMac100 precolumn (300 μm i.d.x5 mm, 100 Å, Thermo Fisher Scientific) using 0.1% formic acid diluted in HPLC grade water. Peptides was further separated using an Easy-Spray RSLC C18 column (75um i.d., 50 cm length, Thermo Fisher Scientific) during a 60-min linear gradient of 0.1% formic acid in acetonitrile (15–35%) at a flow rate of 200 nl min⁻¹. Raw data was acquired in data-dependent acquisition mode (DDA). Full scan mass spectra were acquired in the Orbitrap (Scan range 350–1,500 m/z, resolution 70,000; AGC target, 3⁶, maximum injection time, 50 ms). The 10 most intense peaks were selected for higher-energy collision dissociation (HCD) fragmentation at 30% of normalized collision energy. HCD spectra were acquired in the Orbitrap at resolution 17,500, AGC target 5⁴, maximum injection time 120 ms with fixed mass at 180 m/z. Charge exclusion was selected for unassigned and 1+ ions. The dynamic exclusion was set to 20 s.