Liquid Chromatography Tandem Mass Spectrometry Quantification of 13C-Labeling in Sugars
Abstract
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.
- Authors:
-
[1]
- Univ. of North Texas, Denton, TX (United States)
- The Ohio State Univ., Columbus, OH (United States)
- Publication Date:
- Research Org.:
- Univ. of North Texas, Denton, TX (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1658578
- Grant/Contract Number:
- SC0019233; SC0020325
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Metabolites
- Additional Journal Information:
- Journal Volume: 10; Journal Issue: 1; Journal ID: ISSN 2218-1989
- Publisher:
- MDPI
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; sucrose; glucose 6-phosphate; fructose 6-phosphate; subcellular compartmentation; 13C-labeling; LC-MS/MS; hexokinase; invertase; 13C-metabolic flux analysis
Citation Formats
Cocuron, Jean-Christophe, Ross, Zacchary, and Alonso, Ana P. Liquid Chromatography Tandem Mass Spectrometry Quantification of 13C-Labeling in Sugars. United States: N. p., 2020.
Web. doi:10.3390/metabo10010030.
Cocuron, Jean-Christophe, Ross, Zacchary, & Alonso, Ana P. Liquid Chromatography Tandem Mass Spectrometry Quantification of 13C-Labeling in Sugars. United States. https://doi.org/10.3390/metabo10010030
Cocuron, Jean-Christophe, Ross, Zacchary, and Alonso, Ana P. Fri .
"Liquid Chromatography Tandem Mass Spectrometry Quantification of 13C-Labeling in Sugars". United States. https://doi.org/10.3390/metabo10010030. https://www.osti.gov/servlets/purl/1658578.
@article{osti_1658578,
title = {Liquid Chromatography Tandem Mass Spectrometry Quantification of 13C-Labeling in Sugars},
author = {Cocuron, Jean-Christophe and Ross, Zacchary and Alonso, Ana P.},
abstractNote = {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.},
doi = {10.3390/metabo10010030},
journal = {Metabolites},
number = 1,
volume = 10,
place = {United States},
year = {Fri Jan 10 00:00:00 EST 2020},
month = {Fri Jan 10 00:00:00 EST 2020}
}
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