Liquid Chromatography Tandem Mass Spectrometry Quantification of 13C-Labeling in Sugars

Cocuron, Jean-Christophe; Ross, Zacchary; Alonso, Ana P.

doi:10.3390/metabo10010030

Liquid Chromatography Tandem Mass Spectrometry Quantification of 13C-Labeling in Sugars

Journal Article · Thu Jan 09 23:00:00 EST 2020 · Metabolites

DOI:https://doi.org/10.3390/metabo10010030· OSTI ID:2377023

Cocuron, Jean-Christophe; Ross, Zacchary;

Subcellular compartmentation has been challenging in plant 13C-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 13C-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-13C]-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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Sponsoring Organization:: USDOE

Grant/Contract Number:: SC0019233

OSTI ID:: 2377023

Alternate ID(s):: OSTI ID: 1658578

Journal Information:: Metabolites, Journal Name: Metabolites Journal Issue: 1 Vol. 10; ISSN 2218-1989; ISSN METALU

Publisher:: MDPI AGCopyright Statement

Country of Publication:: Switzerland

Language:: English

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