| MS Description
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- Extraction
Frozen soil sample (300 mg) … - Extraction
Frozen soil sample (300 mg) was extracted with 900 µL of methanol containing 25 µM of 7-hydroxy-5-methylflavone as an internal standard. After homogenizing the samples by mixing twice for 2 min each time using a Mixer Mill MM 300 (QIAGEN K.K., Tokyo, Japan) at 25 Hz, the homogenates were centrifuged (17,400 xg for 5 min at 4C). After recovery of the supernatant from each homogenate, it was filtered through a 0.2-µm polytetrafluoroethylene (PTFE) membrane (Millipore). Hydrophobic compounds in the filtrate were removed by absorption to a C18 silica column (MonoSpin C18, GL Science, Tokyo, Japan). The eluate was used for the untargeted metabolome analysis using LC-MS. Mock samples were prepared as above without adding the soil materials.
- LC-MS
The untargeted metabolome analysis was performed using an Agilent 1100 system (Agilent, Palo Alto, CA) coupled to a Finnigan LTQ-FT (Thermo Fisher Scientific). An aliquot (20 µL) of a methanol solution was applied to a TSK-gel column ODS-100V (4.6 x 250 mm, 5 µm; TOSOH Corporation, Tokyo, Japan). Water (HPLC grade; solvent A) and acetonitrile (HPLC grade; solvent B) were used as the mobile phase with 0.1% v/v formic acid added to both solvents. 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 to 0.25 mL/min (0–100 min) and 0.5 mL/min (100.1–107 min). The column oven temperature was set to 40C. The mass range was m/z 100–1500. The electrospray-ionization (ESI) settings were a spray voltage of 4.0 kV and capillary temperature of 300 °C. The nitrogen sheath gas and auxiliary gas were set at 40 and 15 arbitrary units, respectively. The MSn analysis was performed with ESI positive mode as follows: Full mass scan with FT-ICR at a resolution of 100,000 and a mass scan range 100-1500, and MS2 scans by collision-induced dissociation for the most intense five ions of the full mass scan with ion trap (IT). To monitor the HPLC eluate, a photodiode array detector was used with a wavelength range of 200–650 nm. Data were acquired and browsed using Xcalibur software version 2.0.7 (Thermo Fisher Scientific). version 2.0.7 (Thermo Fisher Scientific).
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