Q exactive orbitrap high resolution mass spectrometer

Dionex UltiMate 3000 high-pressure liquid chromatography (Thermo Fisher Scientific, USA) was used for component separation. An ACQUITY BEH C₁₈ column (2.1 × 50 mm, 1.7 μm, Waters, Milford, MA, USA) was used with 30°C. The mobile phase consisted of acetonitrile with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.21 mL/min. The gradient elution procedure was as follows: 0-0.5min, 7%-10% B; 0.5-5.5min, 10%-15% B; 5.5-6.5min, 15%-15.1% B; 6.5-7.5min, 15.1%-15.2% B; 7.5-9.5min, 15.2%-15.3% B; 9.5-10min, 15.3%-15.5% B; 10-13min, 15.5%-17% B; 13-14min, 17% B; 14-15min, 17%-20% B; 15-17min, 20%-25% B; 17-20min, 25%-28% B; 20-25min, 28%-35% B; 25-26min, 35%-41% B; 26-27.5min, 41%-90% B; 27.5-29.5min, 90%-7% B; and 29.5-30min, 7% B. The injection volume was 0.4 μL. The detection wavelength was 230 nm.
The LC system was coupled to a Q-Exactive Orbitrap High-Resolution Mass Spectrometer (Thermo Fisher Scientific, USA). The mass spectrometer was operated both in positive and negative ion mode with HESI source. The parameters were as follows: N₂; 3.8 KV (positive ion mode) and 3 KV (negative ion mode); ion source temperature 350°C; capillary voltage, 320°C; sheath gas (nitrogen) flow, 40arb; aux gas flow, 10arb; and full-scan spectra mass range, 100~1500 Da.

An Ultimate 3000 UHPLC device (Thermo Fisher, Waltham, MA, USA) equipped with an XBridge BEH C18 column (2.1 × 150 mm, 2.5 μm particle size, Waters, Milford, MA, USA) was coupled with a Q-Exactive Orbitrap high-resolution mass spectrometer (Thermo Fisher) to analyse the peptides. Approximately 10 μg of each sample was injected into the column at an initial condition of 98% mobile phase A (0.1% formic acid in water) and 2% mobile phase B (0.1% formic acid in acetonitrile). The peptides were eluted by first increasing the concentration of mobile phase B to 32% within 42 min and then to 90% mobile phase B within 5 min. The column was washed using 100% mobile phase B and then equilibrated using 2% mobile phase B prior to the next injection. The column oven was set to 45 °C, and the flow rate was 0.2 mL/min. The mass spectrometer was operated in full-scan mode from m/z 200 to 1500 coupled with full MS/dd-MS² in positive-ion mode.