| MS Comment of details
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[column] TSK-gel ODS-100V (4.6 x 250 mm, 5 … [column] TSK-gel ODS-100V (4.6 x 250 mm, 5 micrometer; TOSOH)
[gradient] Solvent A: water containing 0.1% v/v formic acid; Solvent B: acetonitrile containing 0.1% v/v formic acid; Gradient: 3% B (0 min), 97% B (90 min), 97% B (100 min), 3% B (100.1 min), and 3% B (107 min)
[total separation time] 107 min (107 min)
[total separation time] 107 min
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| MS Description
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Metabolite extraction
300 mg of powdered … Metabolite extraction
300 mg of powdered sample was mixed with 900 uL of 100% methanol solution, and 18 mg freeze-dried sample was mixed with 1200 uL of 75% methanol solution in 2 mL tube. Both 100% and 75% methanol solution contain 25 uM of 7-hydroxy-5-methylflavone as internal standard (IS). The sample was homogenized using Mixer Mill MM 300 (QIAGEN) at 25 Hz, for 2 min, twice. The homogenate was centrifuged under 17,400 x g, 5 min at 4 C. A supernatant was passed through a Polytetrafluoroethylene (PTFE) filter (pore size 0.2 um, Millipore), and the filtrate was applied to a C18 silica column (MonoSpin C18, GL Science) to remove highly-hydrophobic contaminants. The filtrate passed through the C18 column was used for LC-MS analyzes.
The same extraction procedure with 75% methanol was performed without a sample to prepare mock samples and used as negative controls.
Liquid chromatography (LC)-mass spectrometry (MS) analysis
Agilent 1100 system (Agilent) and Finnigan LTQ-FT (Thermo Fisher Scientific) were used. Aliquots (5 uL) of the methanol extract were applied to a TSK-gel ODS-100V column (4.6 x 250 mm, 5 um, TOSO), and separated by water containing 0.1%(v/v) formic acid (Solvent A) and acetonitrile containing 0.1%(v/v) formic acid (Solvent B). The gradient program was as follows: 3% B (0 min), 97% B (90 min), 97% B (100 min), 3% B (100.1 min), and 3% B (107 min). The flow rate was set at 0.25 mL/min for 0-100 min, and 0.5 mL/min for 100.1-107 min.
The conditions of mass spectrometry were as below:
- Method 1: This method is used for measuring the accurate mass of the peaks and obtaining MS/MS spectra. [Full scan] resolution, 100,000 by FT; m/z range, 100-1500 [MS2 scan] data-dependent scan by IT for the most intense 5 ions in the full scan. [Dynamic exclusion setting for MS2 scan] repeat count, 3; repeat duration, 30 s; exclusion list size, 500; margin, 10 ppm; exclusion duration, 20 s.
- Method 2: Same as Method 1 except below: [Dynamic exclusion setting for MS2 scan] exclusion duration, 30 s.
- Method 3: Same as Method 1 except below: [Dynamic exclusion setting for MS2 scan] repeat count, 2; exclusion duration, 30 s.
- Method 4: Same as Method 1 except below: [Dynamic exclusion setting for MS2 scan] off.
- Method 5: This method is used for obtaining MS3 spectra. [Full scan] resolution, 12,500 by FT; m/z range, 100-1500. [MS2 scan] data-dependent scan by IT for the most intense 5 ions in the full scan. [Dynamic exclusion setting for MS2 scan] same as Method 1. [MS3 scan] data-dependent scan by IT for the most intense 2 ions in the MS2 scan
- Method 6: This method is used for obtaining MS2 spectra in high-resolution: [Full scan] resolution, 12,500 by FT; m/z range, 100-1500. [MS2 scan] data-dependent scan by FT for the most intense 5 ions in the full scan. resolution, 12,500. [Dynamic exclusion setting for MS2 scan] repeat count, 1; repeat duration, 30 s; exclusion list size, 500; margin, 10 ppm; exclusion duration, 40 s.
- Method 7: Same as Method 5 except below: [Dynamic exclusion setting for MS2 scan] same as Method 2
ESI: electrospray ionization, FT: Fourier transformation ion cyclotron resonance type mass spectrometry, IT: ion trap type mass spectrometry.
The mass analyses with electrospray ionization (ESI) in positive or negative modes were performed. The combinations of the Methods 1-7 and ionization polarity are different between the samples, but, at least one method for accurate mass measurement (Method 1-4) was applied for each sample. The raw data were acquired using Xcalibur software (Thermo Fisher Scientific). libur software (Thermo Fisher Scientific).
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