Silica nano electrospray emitter

Digests (2 μl) were analysed on a 50-cm Easy-Spray column with an internal diameter of 75 μm, packed with 2-μm C18 particles, fused to a silica nano-electrospray emitter (Thermo Fisher Scientific). Reversed phase liquid chromatography was performed using the Ultimate 3000 nano system with a binary buffer system consisting of 0.1% formic acid (buffer A) and 80% acetonitrile in 0.1% formic acid (buffer B). The peptides were separated by a linear gradient of 5%–40% buffer B over 110 minutes at a flow rate of 300 nl/min. The column was operated at a constant temperature of 35°C, and the LC system coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific). The Q-Exactive was operated in data-dependent mode with survey scans acquired at a resolution of 70 000 at m/z 200. Up to the top 10 most abundant isotope patterns with charge states +2, +3, and/or +4 from the survey scan were selected with an isolation window of 2.0 Th and fragmented by higher-energy collisional dissociation with normalized collision energies of 30. The maximum ion injection times for the survey scan and the MS/MS scans were 250 and 100 ms, respectively, and the ion target value was set to 1E6 for survey scans and 1E4 for the MS/MS scans. Repetitive sequencing of peptides was minimized through dynamic exclusion of the sequenced peptides for 20 seconds.

Peptides were analysed by on-line nanoflow liquid chromatography (LC) using the Thermo EASY-nLC 1000 LC system (Thermo Fisher Scientific) coupled with Q-Exactive mass spectrometer (Thermo Fisher Scientific). Samples were loaded onto an Easy-Spray C₁₈ column (50 cm, inner diameter 75 µm), fused to a silica nano-electrospray emitter (Thermo Fisher Scientific). Chromatography was performed at 35°C with a buffer system consisting of 0.1% formic acid (buffer A) and 80% acetonitrile in 0.1% formic acid (buffer B). The peptides were separated over a 97 min linear gradient of 3.8-50% buffer B at a flow rate of 300 nl/min. The Q-Exactive was operated in data-dependent mode with dynamic exclusion and survey scans acquired at a resolution of 70,000. The 10 most abundant isotope patterns with charge states +2, +3 and/or +4 from the survey scan were selected with an isolation window of 2.0 Th and fragmented by higher energy collisional dissociation with normalised collision energies of 30. The maximum ion injection times for the survey scan and the MS/MS scans were 250 and 100 ms, respectively, and the ion target value was set to 1E6 for survey scans and 1E4 for the MS/MS scans.