Nano lc 1200

LC–MS/MS instrumentation consisted of a nano-LC 1200 (Thermo Fisher Scientific) coupled via a nano-electrospray ionization source to a quadrupole-based Q Exactive HF-x instrument.
Peptides were separated on in-house packed 50-cm columns (1.8 µm C18 ReproSil Dr Maisch) within a total gradient length of 90 min. The column temperature was controlled by an in-house-designed column oven at 50 °C. The flow rate was 250 nl min⁻¹. The spray settings were: 2.4 kV; capillary temperature, 275 °C; no auxiliary gas applied.
The mass spectrometer operated in a data-dependent mode targeting the top 22 intense peaks for fragmentation and MS/MS spectra acquisition. MS/MS spectra were acquired at 17,500 resolution (200 m/z) using an isolation window of 1.4 Th. The maximum injection time was set to 22 ms. The normalized collision energy was set to 29 and dynamic exclusion was enabled for 20 s.

All peptide samples were measured in a single-shot manner in a Q-Exactive HF-X hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) after peptide separation by high-performance liquid chromatography (nanoLC 1200, Thermo Fisher Scientific) using a 50 cm reversed-phase column (made in house, packed with 1.9 µm C18 ReproSil particles). Peptides were eluted over a 90-minute-gradient from 0% to 95% buffer B (0.1% formic acid and 80% ACN) with a flow rate of 300 nL/minute.
Full scans were obtained from 300 to 1650 m/z with a target value of 3 × 10⁶ ions at a resolution of 60,000 at 200 m/z. The fifteen most intense ions (Top15) of each full scan were fragmented with higher-energy collisional dissociation (HCD) (target value 1 × 10⁵ ions, maximum injection time 120 ms, isolation window 1.4 m/z, underfill ratio 1%), and fragments were detected in the Orbitrap mass analyzer at a resolution of 15,000 at 200 m/z.