| MS Description
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Sample extraction<br />
Crushed and … Sample extraction<br />
Crushed and frozen samples (10 g) were resuspended in 50 mL of MeOH, including internal standards for standardization of peak areas, and centrifuged (16,000 × g, 3 min, 4°C). Supernatants were dispensed for each analysis as follows: 0.5 mL for ionic metabolites, 1 mL for neutral metabolites, 1 mL for sugars, and 47.5 mL for plant hormones. Because of the small amounts of pear fruit samples obtained in the early periods (2WBB, 1WBB, and B), the initial amounts of these samples were 1 g, extracted in 20 mL of MeOH, and were separately used for each analysis as described above. The residues were subsequently used for starch analysis.<br />
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Quantification of plant hormones using LC-tripleQ MS<br />
Fifteen plant hormones were detected and quantified using stable isotopes of each hormone and LC-tripleQ MS analyses were performed as previously reported. Briefly, methanol extracts were evaporated and resuspended in 5 volumes of 80% MeCNaq. containing 1% AcOH with internal standards and deuterated plant hormones, for quantification, and then extracted by mixing occasionally on ice for 1 h. After centrifugation, the pellet was resuspended and extracted with the same volume of the solvent described above, and supernatants were mixed. The MeCN was evaporated and the residual solution was purified using solid-phase extraction columns. Brassinosteroids required further purification by HPLC. ids required further purification by HPLC.
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