| MS Description
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<Sample processing and extraction>&l … <Sample processing and extraction><br />
The lyophilized sample in a 2 ml tube was frozen and then homogenized with a 5 mm of zirconia bead by a Mixer Mill (Retsch, Haan, Germany) at 20 Hz for 1 min. Five mg dry weight (DW) of the lyophilized samples were weighed for GC-MS and LC-MS analyses, while 25 mg DW of the samples for CE-MS analysis.<br /><br />
<Extraction and derivatization for GC-MS><br />
Each sample was extracted with a concentration
of 2.5 mg DW of tissues per ml extraction medium (methanol / chloroform/water [3:1:1 v/v/v])
containing 10 stable isotope reference compounds:<br />
[2H4]-succinic acid,<br />
[13C5,15N]-glutamic acid,<br />
[2H7]-cholesterol,<br />
[13C3]-myristic acid,<br />
[13C5]-proline,<br />
[13C12]-sucrose,<br />
[13C4]-hexadecanoic acid,<br />
[2H4]-1,4-butanediamine,<br />
[2H6]-2-hydoxybenzoic acid and<br />
[13C6]-glucose<br />
using a Retsch mixer mill MM310 at a frequency of 30 Hz for 3 min at 4℃. Each isotope compound
was adjusted to a final concentration of 15 ng µl-1 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). For methoximation, 30
µl of methoxyamine hydrochloride (20 mg/ml in pyridine) was added to the sample. After 24 h of
derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 µl of MSTFA
with 1% TMCS at 37℃ 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.<br />
Helium was used as the carrier gas at a constant flow rate of 1 ml min-1. The temperature
program for metabolome analysis started with a 2-min isothermal step at 80 ℃ and this was
followed by temperature ramping at 30 ℃ to a final temperature of 320 ℃, which was maintained
for 3.5 min. The transfer line and the ion source temperatures were 250 and 200 ℃, 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 273 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-1 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) (Wagner et al. 2003; Schauer et al. 2005). The normalized response
for the calculation of the signal intensity of each metabolite from the mass-detector response
was obtained by each selected ion current that was unique in each metabolite MS spectrum to
normalize the peak response. For quality control, we injected methylstearate in every 6 samples.
Data was normalized using the CCMN algorithm (Redestig et al. 2009). the CCMN algorithm (Redestig et al. 2009).
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