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Sample preparation
Total lipids were extracted according to the method of Bligh and Dyer (Bligh and Dyer 1959) with slight modifications. The frozen plant material was milled on a Mixer Mill MM300 (Retsch, Haan, Germany) for 2 min at 15 Hz with a zirconium bead. The milling process was carried out under cryogenic conditions (the grinding jars were cooled with liquid nitrogen before each milling process to ensure a low milling temperature to keep the plants frozen). The frozen powdered plant material was kept in liquid nitrogen and a 20-fold volume of extraction solvent (CHCl3–MeOH–H2O = 50:100:31.45, v/v/v) was added with 1 μM 10:0/10:0 PC as the internal standard, followed by vigorous stirring on a vortex mixer. After dark incubation for 5 min at room temperature, the homogenate was centrifuged at 3,000×g for 3 min at room temperature. Two hundred microliters of supernatant was mixed with CHCl3 and H2O (52.6 μl each), stirred on a vortex mixer, and incubated on ice for 15 min in darkness. The mixture was centrifuged at 1,000×g for 3 min, and 85 μl of the lower layer was collected. The organic phase was dried by a centrifugal concentrator at room temperature and the residue was dissolved in 81 μl of CHCl3 for LC-MS analysis.
metadata (from Supplementary material 1)
Chromatography instrument description: Shimadzu, LC-20AD HPLC system, operated by LCMSsolution v.3.6.
Auto-injector: Shimadzu, SIL-20AC, LCMSsolution v.3.6., 1microL injection, 1 wash per injection (0.2 mL MeCN)
Separation column and pre/guard column: Shimadzu, Atlantis HILIC Silica (pore size: 3 microm) 2.1 by 100 mm
Separation parameter: A two-solvent system was used to generate the mobile phase: solvent A, methanol-water (95:5, v/v) containing 0.2% ammonium formate (pH 5.9); solvent B, acetonitrile-methanol-water (95:2:3, v/v/v) containing 0.2% ammonium formate (pH 5.9). The pH of both solvents A and B was adjusted by adding 30% NH4OH to the mixtures of solvents containing 0.2% (v/v) formic acid.; elution program: 100% solvent B (0-3.33 min), 100~65% solvent B (3.33-10 min), 65~30% (10-11.33 min), 30% solvent B (11.33-14.66 min), 100% solvent B (14.66-40 min). The flow rate was held at 0.2 mL/min, and it was then increased to 0.4 mL/min at 14.66 min after the beginning of the gradient, maintained for 13.33 min, and then decreased to 0.2 mL/min. Total elution time was 40 min.
Instrument description: Shimadzu, LCMS-IT-TOF, operated by LCMSsolution v.3.6.
Sample introduction and delivery: From LC
Ionization source: ESI negative. interface voltage, 4.5 V; curved desolvation line temperature, 200C; heat block temperature, 200C; ion accumulation time, 10 msec; nebulizer gas, N2 (15 L/min).
Mass analyzer description and acquisition mode: ion trap-time-of-flight operated at scan mode
Data acquisition parameters: detection mode: scan (m/z 150-1600; event time: 0.2 s; profile).
Data file format: Original data file (.lcd)
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