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<Extraction and Derivatization for GC-T … <Extraction and Derivatization for GC-TOF-MS><br />
Each sample was extracted with a concentration of 2.5 mg dry weight (DW) of tissues per ml 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. These internal standards were used to normalize the data using cross-contribution compensating mutipl standard normalization (CCMN).
Each isotope compound was adjusted to a final concentration of 15 ng/μL for each 1 μL injection. After centrifugation, a 200 μL aliquot of the supernatant (∼0.5 mg of DW of each sample) was drawn and transferred into a glass insert vial for a pilot experiment. We mixed leaf extracts (at the second internode of the second truss) and fruit extracts (mixture of pericarp and jelly/seed) for a gradient experiment. The percentages of leaf:fruit mixture extracts are given in Supporting Information Table 1. <br />
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 derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 L MSTFA (Tokyo Chemical Industry, Tokyo, Japan) at 37 ℃ with shaking. A 30 μL aliquot of n-heptane was added following silylation. All derivatization steps were performed in a VSC-100 vacuumglovebox (Sanplatec, Japan) filled with 99.9995% (G3 grade) dry nitrogen.<br /><br />
<GC-TOF-MS Conditions><br />
For metabolome analysis, 1 μL of extracts (∼5.6 μg of each sample) was injected in the splitless mode by a CTC CombiPAL autosampler (CTC Analytics, Zwingen, Switzerland) into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmington, DE, 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, PA, USA).
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 237 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 rangeof a mass-to-charge ratio of 60 ≦ m/z ≦ 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). 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
spectrumto normalize the peak response. For quality control, we injected methylstearate into every sixth sample. ed methylstearate into every sixth sample.
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