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Plant Metabolic Flux Analysis

Volume 1090 of the series Methods in Molecular Biology pp 121-130

Date:

Quantifying 13C-labeling in Free Sugars and Starch by GC-MS

  • Mohamed KoubaaAffiliated withDepartment of Molecular Genetics, The Ohio State University
  • , Brigitte ThomassetAffiliated withUniversite de Technologie de Compiegne, CNRS-FRE 3580, Gènie Enzymatique et Cellulaire, Centre de Recherche de Royallieu
  • , Albrecht RoscherAffiliated withGénie Enzymatique et Cellulaire, FRE CNRS 3580, Université de Picardie

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Abstract

We describe an approach to extract 13C-labeled sugars (glucose, fructose, maltose, sucrose, myo-inositol as well as glucose from starch) from plant tissues and to analyze their isotopomer distribution by gas chromatography–mass spectrometry (GC-MS). Sugars are derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) into their Si(CH3)3 derivatives. Electronic and chemical ionizations are used to obtain suitable fragments for metabolic flux analysis (MFA). Unique fragments are identified by computer simulation and experimental verification with labeled standards. Linear equations for separating information from glucosyl and fructosyl moieties of sucrose are presented. Finally, mass distributions are corrected for natural isotope abundance using a home-written program. The method is illustrated by sugar isotopomer analysis of 13C-labeled rapeseed embryos.

Key words

Metabolic flux analysis Isotopomers GC-MS 13C-labeling Sugars Brassica napus