| MS Description
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BioSource amount:<br />
The harveste … BioSource amount:<br />
The harvested samples were weighed and then each biological sample was put in a 2-ml tube with 5 mm Zirconia beads to be used for metabolite profiling.<br /><br />
For GC-TOF-MS analysis:<br />an equivalence of 6 μg fresh weight (FW) of the derivatised sample was injected.<br /><br />
Sample processing and extraction: <br />The frozen sample in a 2 ml tube was extracted with a concentration of 25 mg FW of tissues per ml extraction medium (methanol/chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds using a Retsch mixer mill MM310 (Retsch, Haan, Germany) at a frequency of 30 Hz for 3 min at 4°C. After centrifugation, a 200-μl of the supernatant was used for GC-TOF-MS analysis, while a 100-μl of the supernatant for LC-q-TOF-MS analysis.<br /><br />
Extraction and derivatization for GC-TOF-MS analysis:<br />Each sample was extracted with a concentration of 25 mg FW of tissues per µl extraction medium (methanol/chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds ([2H4]-succinic acid, [13C5,15N]-glutamic acid, [2H7]-cholesterol, [13C3]-myristic acid, [13C5]-proline, [13C12]-sucrose, [13C4]-hexadecanoic acid, [2H4]-1,4-butanediamine, [2H6]-2-hydoxybenzoic acid, and [13C6]-glucose) using a Retsch mixer mill MM310 at a frequency of 30 Hz for 3 min at 4°C. Each isotope compound was adjusted to a final concentration of 15 ng/µl for each 1-µl injection. After centrifugation for 5 min at 15,100 × g, a 200-µl aliquot of the supernatant was drawn and transferred into a glass insert vial. The extracts were evaporated to dryness in an SPD2010 SpeedVac® concentrator from ThermoSavant (Thermo electron corporation, Waltham, MA, USA).<br />
For methoximation, 30 µl of methoxyamine hydrochloride (20 mg/ml in pyridine) was added to the sample. After 24h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 µl of MSTFA with 1% TMCS at 37°C with shaking. Thirty µl of n-heptane was added following silylation. All the derivatization steps were performed in the vacuum glove box VSC-100 (Sanplatec, Japan) filled with 99.9995% (G3 grade) of dry nitrogen.<br /><br />
GC-TOF-MS conditions<br />
One microliter of each sample was injected in the splitless mode by an CTC CombiPAL autosampler (CTC analytics, Zwin-gen, Switzerland) into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, USA) equipped with a 30 m × 0.25 mm inner diameter fused-silica capillary column with a chemically bound 0.25-μl film Rtx-5 Sil MS stationary phase (RESTEK, Bellefonte, USA) for metabolome analysis. Helium was used as the carrier gas at a constant flow rate of 1 ml/min. The temperature program for metabolome analysis started with a 2-min isothermal step at 80 °C and this was followed by temperature ramping at 30 °C to a final temperature of 320 °C, which was maintained for 3.5 min. The transfer line and the ion source temperatures were 250 and 200 °C, respectively. Ions were generated by a 70-eV electron beam at an ionization current of 2.0 mA. The acceleration voltage was turned on after a solvent delay of 235 s. Data acquisition was performed on a Pegasus IV TOF mass spectrometer (LECO, St. Joseph, MI, USA) with an acquisition rate of 30 spectra/s in the mass range of a mass-to-charge ratio of m/z = 60–800. Alkane standard mixtures (C8–C20 and C21–C40) were purchased from Sigma–Aldrich (Tokyo, Japan) and were used for calculating the retention index (RI) (Schauer et al., 2005, Wagner et al., 2003). For quality control, we injected methylstearate in every 6 samples. njected methylstearate in every 6 samples.
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