| MS Description
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Total lipids were extracted using a mixtur … Total lipids were extracted using a mixture of methyl tert-butyl ether (MTBE) and methanol, as previously reported (Matyash et al. 2008, Giavalisco et al. 2011), with slight modifications. The frozen plant samples were milled to a fine powder under cryogenic conditions, as described previously (Okazaki et al. 2013a).
Total lipids were extracted by adding 16 volumes of extraction solvent (3:1, MTBE/methanol [v/v] with 1 μM 1,2-didecanoyl-sn-glycero-3-phosphocholine [Sigma-Aldrich] added as an internal standard) to the powdered plant sample while in liquid nitrogen, followed by vigorous vortexing. To this mixture, four volumes of water was added, followed by vigorous mixing on a sample tube mixer for 5 min at room temperature, incubation on ice for 15 min, and centrifugation at 1000 g at 5°C for 5 min. After centrifugation, a 100-μl aliquot of the supernatant was collected and evaporated to dryness in a centrifugal concentrator. The residue was reconstituted in 125 μl of ethanol and centrifuged at 10 000 g at 5°C for 15min. The supernatant was used immediately for lipid analysis.<br />
Lipid analysis was conducted using liquid chromatography quadrupole time-of-flight mass spectrometry on a Waters Xevo G2 Q-TOF MS combined with a Waters ACQUITY UPLC system, as reported previously (Kimbara et al. 2013). The conditions for recording MS/MS in positive-ion mode were as follows: scan range, m/z 100–1200; capillary, 3.0 kV; sampling cone, 20; extraction cone, 4.0; source temperature, 120°C; desolvation temperature, 450°C; cone gas flow, 50 l h−1; desolvation gas flow, 600 l h−1; collision energy ramp start, 20 eV; collision energy ramp end, 50 eV. The conditions for recording MS/MS in negative-ion mode were as follows: scan range, m/z 100–1200; capillary, 2.5 kV; sampling scone, 40; extraction cone, 4.0; source temperature, 120°C; desolvation temperature, 450°C; cone gas flow, 50 l h−1; desolvation gas flow, 600 l h−1; collision energy ramp start, 20 eV; and collision energy ramp end, 50 eV.<br />
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Detection of GlcADG in tomato and soybean<br />
Ten-day-old tomato and soybean seedlings were transplanted to rockwool, and nutrients were supplied by the application of liquid nutrient medium. For the P-limitation treatment, liquid medium without potassium phosphate was used. After a 14-day incubation period, lipids were extracted from the leaves and analyzed by LC-Q-TOF-MS. om the leaves and analyzed by LC-Q-TOF-MS.
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