Journal
METABOLITES
Volume 10, Issue 1, Pages -Publisher
MDPI
DOI: 10.3390/metabo10010030
Keywords
sucrose; glucose 6-phosphate; fructose 6-phosphate; subcellular compartmentation; C-13-labeling; LC-MS/MS; hexokinase; invertase; C-13-metabolic flux analysis
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Funding
- Agriculture and Food Research Initiative competitive grant from the USDA National Institute of Food and Agriculture [2016-67013-29020]
- U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (BER) [DE-SC0019233]
- Genomic Science Program grant [DE-SC0020325]
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Subcellular compartmentation has been challenging in plant C-13-metabolic flux analysis. Indeed, plant cells are highly compartmented: they contain vacuoles and plastids in addition to the regular organelles found in other eukaryotes. The distinction of reactions between compartments is possible when metabolites are synthesized in a particular compartment or by a unique pathway. Sucrose is an example of such a metabolite: it is specifically produced in the cytosol from glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). Therefore, determining the C-13-labeling in the fructosyl and glucosyl moieties of sucrose directly informs about the labeling of cytosolic F6P and G6P, respectively. To date, the most commonly used method to monitor sucrose labeling is by nuclear magnetic resonance, which requires substantial amounts of biological sample. This study describes a new methodology that accurately measures the labeling in free sugars using liquid chromatography tandem mass spectrometry (LC-MS/MS). For this purpose, maize embryos were pulsed with [U-C-13]-fructose, intracellular sugars were extracted, and their time-course labeling was analyzed by LC-MS/MS. Additionally, extracts were enzymatically treated with hexokinase to remove the soluble hexoses, and then invertase to cleave sucrose into fructose and glucose. Finally, the labeling in the glucosyl and fructosyl moieties of sucrose was determined by LC-MS/MS.
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