Liquid Chromatography Tandem Mass Spectrometry for Measuring 13C-labeling in Intermediates of the Glycolysis and Pentose Phosphate Pathway

2013 ◽  
pp. 131-142 ◽  
Author(s):  
Jean-Christophe Cocuron ◽  
Ana Paula Alonso
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Pentose Phosphate Pathway ◽  
Pentose Phosphate ◽  
Tandem Mass ◽  
13C Labeling ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

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

Metabolites ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 30 ◽  
Author(s):  
Jean-Christophe Cocuron ◽  
Zacchary Ross ◽  
Ana P. Alonso
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Time Course ◽  
Metabolic Flux ◽  
Flux Analysis ◽  
Tandem Mass ◽  
13C Labeling ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

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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