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

被引:6
作者
Cocuron, Jean-Christophe [1 ]
Ross, Zacchary [2 ]
Alonso, Ana P. [1 ,3 ]
机构
[1] Univ North Texas, BioDiscovery Inst, Denton, TX 76203 USA
[2] Ohio Univ, Heritage Coll Osteopath Med, Dublin, OH 43016 USA
[3] Univ North Texas, Dept Biol Sci, Denton, TX 76203 USA
基金
美国食品与农业研究所;
关键词
sucrose; glucose; 6-phosphate; fructose; subcellular compartmentation; C-13-labeling; LC-MS/MS; hexokinase; invertase; C-13-metabolic flux analysis; METABOLIC FLUX ANALYSIS; TRICARBOXYLIC-ACID CYCLE; MAIZE ROOT-TIPS; STEADY-STATE; COMPARTMENTATION; PATHWAYS; TUBERS; CELLS; SEEDS;
D O I
10.3390/metabo10010030
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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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页数:11
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