SE58 > SS01
ID SE58_SS01
Title Sample processing and extraction
Description 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.

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.
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