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Title: 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];  [2]; ORCiD logo [1]
  1. Univ. of North Texas, Denton, TX (United States)
  2. 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. https://doi.org/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 = {2020},
month = {1}
}

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Works referenced in this record:

Measuring multiple fluxes through plant metabolic networks
journal, February 2006


Fluxes of carbohydrate metabolism in ripening bananas
journal, October 1993


Compartmentation of metabolism within mitochondria and plastids
journal, April 2001


Compartmentation in plant metabolism
journal, June 2006


Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry
journal, May 2010

  • Alonso, Ana P.; Piasecki, Rebecca J.; Wang, Yan
  • Plant Physiology, Vol. 153, Issue 3
  • DOI: 10.1104/pp.110.155713

Compartment-specific labeling information in 13C metabolic flux analysis of plants
journal, August 2007


Understanding fatty acid synthesis in developing maize embryos using metabolic flux analysis
journal, September 2010


Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos: Metabolic fluxes in sunflower embryos
journal, August 2007


A metabolic flux analysis to study the role of sucrose synthase in the regulation of the carbon partitioning in central metabolism in maize root tips
journal, September 2007


Substrate cycles in the central metabolism of maize root tips under hypoxia
journal, August 2007


Central metabolic fluxes in the endosperm of developing maize seeds and their implications for metabolic engineering
journal, January 2011


A Combined Metabolomics and Fluxomics Analysis Identifies Steps Limiting Oil Synthesis in Maize Embryos
journal, September 2019

  • Cocuron, Jean-Christophe; Koubaa, Mohamed; Kimmelfield, Rebecca
  • Plant Physiology, Vol. 181, Issue 3
  • DOI: 10.1104/pp.19.00920

Quantification of Compartmented Metabolic Fluxes in Maize Root Tips Using Isotope Distribution from 13 C- or 14 C-Labeled Glucose
journal, June 1995

  • Dieuaide-Noubhani, Martine; Raffard, Gérard; Canioni, Paul
  • Journal of Biological Chemistry, Vol. 270, Issue 22
  • DOI: 10.1074/jbc.270.22.13147

Subcellular Flux Analysis of Central Metabolism in a Heterotrophic Arabidopsis Cell Suspension Using Steady-State Stable Isotope Labeling
journal, November 2009

  • Masakapalli, Shyam K.; Le Lay, Pascaline; Huddleston, Joanna E.
  • Plant Physiology, Vol. 152, Issue 2
  • DOI: 10.1104/pp.109.151316

The Metabolic Architecture of Plant Cells: STABILITY OF CENTRAL METABOLISM AND FLEXIBILITY OF ANABOLIC PATHWAYS DURING THE GROWTH CYCLE OF TOMATO CELLS
journal, September 2002

  • Rontein, Denis; Dieuaide-Noubhani, Martine; Dufourc, Erick J.
  • Journal of Biological Chemistry, Vol. 277, Issue 46
  • DOI: 10.1074/jbc.M206366200

13C labeling analysis of sugars by high resolution-mass spectrometry for metabolic flux analysis
journal, June 2017

  • Acket, Sébastien; Degournay, Anthony; Merlier, Franck
  • Analytical Biochemistry, Vol. 527
  • DOI: 10.1016/j.ab.2017.02.005

Targeted Metabolomics of Physaria fendleri, an Industrial Crop Producing Hydroxy Fatty Acids
journal, February 2014

  • Cocuron, Jean-Christophe; Anderson, Brooke; Boyd, Alison
  • Plant and Cell Physiology, Vol. 55, Issue 3
  • DOI: 10.1093/pcp/pcu011

Structural Characterization of Neutral Saccharides by Negative Ion MALDI Mass Spectrometry Using a Superbasic Proton Sponge as Deprotonating Matrix
journal, May 2017

  • Calvano, Cosima Damiana; Cataldi, Tommaso R. I.; Kögel, Julius F.
  • Journal of The American Society for Mass Spectrometry, Vol. 28, Issue 8
  • DOI: 10.1007/s13361-017-1679-y

Highlighting the tricarboxylic acid cycle: Liquid and gas chromatography–mass spectrometry analyses of 13C-labeled organic acids
journal, May 2013

  • Koubaa, Mohamed; Cocuron, Jean-Christophe; Thomasset, Brigitte
  • Analytical Biochemistry, Vol. 436, Issue 2
  • DOI: 10.1016/j.ab.2013.01.027

High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry
journal, March 2017