Q exactive hybrid quadrupole orbitrap mass spectrometry

Chromatographic separation of all samples was performed using a ThermoFisher Ultimate 3000 UHPLC system with a Waters ACQUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm). Mobile phases were solvent A (water containing 15 mM ammonium acetate (pH = 9)) and solvent B (90% acetonitrile containing 10 mM ammonium acetate). Linear gradient elution was performed as follows: 0 min, 90% B; 4 min, 85% B; 11-18 min, 75% B; 18.1-20 min, 90% B. Flow rate was 0.25 mL/min. Eluents were analyzed on a ThermoFisher Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometry in heated electrospray ionization positive and negative modes, separately. The main parameters were set as follows: (1) spray voltage, 3500 V; (2) capillary and probe heater temperature, both 350°C; (3) sheath gas flow rate, aux gas flow rate, and S-Lens RF level, 40 (Arb), 10 (Arb), and 50 (Arb), respectively; (4) full scan was operated at high-resolution (35000 FWHM, m/z = 200), range 70-1050 m/z, with AGC target, 3 × 10⁶. Data-dependent acquisition was used to acquire fragment ion information from up to 8 precursors in each scan. Parameters were as follows: HCD energy, 15, 30, and 45 eV; mass resolution, 17500 FWHM; and AGC threshold, 2 × 10⁵.

The sample dissolved in 0.1% FA was analyzed in an Ultimate 3000 UPLC system (Thermo Scientific, Waltham, MA, USA) using a pre-column of Acclaim PepMap RPLC C₁₈ column (300 μm × 5 mm, 100 Å, 5 μm; Thermo Scientific) and an analytical nano-column of Acclaim PepMap RPLC C₁₈ column (150 μm × 150 mm, 100 Å, 1.9 μm; Thermo Scientific). Gradient elution was used with a mobile A of 0.1% FA in 2% ACN and a mobile B of 0.1% FA in 80% ACN. Mobile B increased from 6% to 9% in 5 min, 9% to 14% in 15 min, 14% to 30% in 30 min, 30% to 40% in 8 min, and 40% to 95% in 2 min. The flow rate was set at 600 nL/min. The sample was then analyzed using Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometry (Thermo Scientific). The spray voltage and the capillary temperature were set at +2.2 kV and 270 °C. The m/z for the full scan was set from 100 to 1200. The scan resolutions for MS and MS/MS were set to 70,000 and 17,500, respectively. The normalized collision energy setting was set at 40. The raw MS data file was analyzed and searched against the database of UniProt Avena sativa L. (Oat) using Byonic. The precursor ion mass tolerance and MS/MS tolerance were set at 20 ppm and 0.02 Da, respectively. Only highly confidently recognized peptides were used.