Quantifying 13C-labeling in Free Sugars and Starch by GC-MS
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 wordsMetabolic flux analysis Isotopomers GC-MS 13C-labeling Sugars Brassica napus
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