Q exactive plus orbitrap ms

The procedures of LC/MS-MS were performed from [19 (link)]. Soybean proteins (150 μg) were dissolved with 100 μL of 50 mM NH₄HCO₃ solution, reduced by 2 μL of 500 mM dithiothreitol and then alkylated by 14 μL of 500 mM iodoacetamide. After the resulting samples were filtered by 10-kDa cutoff filter and washed twice with pH 8.5 buffer solution (8 M urea in 100 mM Tris/HCl), the retentate sample was dissolved with 100 μL of 50 mM NH₄HCO₃ solution and then digested with trypsin at 37 °C for overnight. An 8 μL of C₂HF₃O₂ solution (10%, v/v) was added to end the digested reaction. The resulting peptides were desalted by C18 tips, then vacuum dried. The dried samples were dissolved with 20 μL of 0.1% formic acid solution. Four microliters of the resuspended sample were injected into a C18 pre-Column (5 μm, 100 μm × 2 cm). Then, the sample was separated on a C18 analytical column (3 μm, 75 μm × 100 nm) with a linear gradient of mobile phase (0.1% formic acid in 80% acetonitrile). The flow rate was 300 nL/min. After that, the resulting sample was analyzed by Q ExactivePlus-Orbitrap MS (Thermo Fisher Scientific) at a range of 350–2000 m/z, 1.8 kV spray voltage and 300 °C heater temperature. MS/MS was conducted at a range of 200–2000 m/z, 27 eV collision energy and 60 s dynamic exclusion.

Chromatographic analysis was performed on Vanquish UHPLC (Thermo Scientific, Karlsruhe, Germany) with a Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm). The flow rate was 0.3 mL/min. The column temperature was 35 °C. The injection volume was 3 μL. For metabolite separation, the mobile phase (A) consisted of acetonitrile, and the mobile phase (B) consisted of 0.1% formic acid-water. The gradient method followed the steps: 0–5.0 min, 95–70% B; 5.0–10.0 min, 70–50% B; 10.0–27.0 min, 50–10% B; 27.0–27.1 min, 10–95% B; 27.1–30.0 min, 95% B.
The Q-Exactive Plus Orbitrap MS (Thermo Scientific, Dreieich, Germany) was equipped with a heated electrospray ionization source (HESI). The high-resolution scanning range was m/z 80–1200 at a resolution of 70,000 with AGC target at 3 × 10⁶. The dd-MS² data were obtained at a resolution of 17,500 with AGC target at 1 × 10⁶. The detection modes were positive ion and negative ion. Nitrogen (purity ≥ 99.99%) was used as sheath gas and auxiliary gas, and the flow rate was 45 and 10 (arbitrary units). Capillary temperature was 320 °C, and evaporator temperature was 400 °C. Spray voltage was 3800/3500 V (+/−). Probe heater temperature was 320 °C. The stepped normalized collision energy (NCE) was set at 15, 30, and 45. S-Lens RF Level was 50.00.

The MS analysis was performed on Q-Exactive Plus Orbitrap MS (Thermo Scientific, Germany) with a heated electrospray ionization (HESI) source. A high-resolution mass spectrum was acquired at full scan in a mass range of m/z 80–m/z 1,200 at a resolution of 70,000 with AGC target at 3e⁶. The dd-MS² data were obtained at a resolution of 17,500 with AGC target at 1e⁶. Mass spectrometric detection was performed in positive and negative ion modes. The ion source parameters were set as follows: sheath gas and auxiliary gas of nitrogen (purity ≥99.99%) with the flow rate of 45 arb and 10 arb; capillary temperature of 320 °C; spray voltage of 3,800/3,500 V (+/-); probe heater temperature of 320°C. The stepped normalized collision energy (NCE) was set at 15, 30 and 45. S-Lens RF Level was 50.00.