| MS Description
|
- Compound Extraction
Approximately 10 m … - Compound Extraction
Approximately 10 mg of dried fine powder of soybean leaves was accurately weighed in a 2 mL tube, and a 40 times volume of 75% methanol, containing 1 μM of 7-hydroxy-5-methylflavone as an internal standard, was added. Then, the metabolite extraction and the metabolome analysis were performed according to previously described procedures using LC (Nexera X2, Shimadzu), time-of-flight MS (Compact, Bruker), and C18 column (InertSustain AQ-C18, 3 μm x 2.1 mm x 150 mm, GL Sciences) in both ESI positive and negative modes (Sakurai et al., Nucleic Acids Res 51(D1): D660-677, 2023).
- Liquid chromatography (LC)-mass spectrometry (MS) analysis
LC-MS anaysis was conducted as previously described (Sakurai et al., Nucleic Acids Res 51(D1): D660-677, 2023) using Nexera X2 system (Shimadzu Corporation) and Compact system (Bruker Japan K.K.). An aliquot (2 uL) of the methanol extract was applied to an InertSustain AQ-C18 column (3 um x 2.1 mm x 150 mm, GL Sciences) connected after a guard column (InertSustain AQ-C18 Cartridge Guard Column E, GL Sciences), and separated by water containing 0.1%(v/v) formic acid (Solvent A) and acetonitrile (Solvent B). The gradient program was as follows: 2% B (0 min), 2% B (3 min), 98% B (30 min), 98% B (35 min), 2% B (35.01 min), and 2% B (42 min). The flow rate was set at 0.2 mL/min. The column oven temperature was set to 40 degree C.
The compounds separated by the LC were detected using the mass spectrometer under the conditions below: Ionization, Electrospray Ionization (ESI); Polarity, Positive; Scan rate, 1 Hz; Mass scan range, 50-1200; End plate offset, 500 V; Capillary voltage, 4000 V; Nebulizer gas (N2) pressure, 2.5 bar; Dry gas (N2) flow, 8.0 L/min; Dry gas temperature, 200 degree C; Transfer Funnel1 RF, 200.0 Vpp; Funnel2 RF, 200.0 Vpp; In-source CID Energy, 0.0 eV; Hexapole RF, 50.0 Vpp; Quadrupole Ion Energy, 3.0 eV; Low mass m/z, 55.00; Collision energy, 10.0 eV; Collision RF, 450.0 Vpp; Transfer time, 80.0 us; and PrePulse storage, 3.0 us. The data-dependent MS/MS spectra were obtained with the conditions below: Isolation width, 3-15 Da; Collision energy 35 eV; Precursor ion number, 5; Active Exclusion, on; Exclude, after 3 spectra; Release, after 0.2 min; Reconsider precursor, on; and if current intens. / previous intens., 2.0. For mass calibration, 1 mM sodium formate in 50% (v/v) 2-propanol was injected directly into the MS at 38.50–40.50 min of LC separation with a flow rate 0.1 mL/min. The eluent at 0-3 min was wasted. The raw data were obtained by Hystar software (ver.3.2 SR4, Bruker Daltonik, GmbH). ware (ver.3.2 SR4, Bruker Daltonik, GmbH).
|
