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

  • Mohamed Koubaa
  • Brigitte Thomasset
  • Albrecht Roscher
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1090)

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 

References

  1. 1.
    Koubaa M, Cocuron JC, Thomasset B, Alonso AP (2013) Highlighting the tricarboxylic acid cycle: liquid and gas chromatography–mass spectrometry analyses of 13C-labeled organic acids. Anal Biochem 436:151–159PubMedCrossRefGoogle Scholar
  2. 2.
    Koubaa M, Mgaieth S, Thomasset B, Roscher A (2012) Gas chromatography–mass spectrometry analysis of 13C labeling in sugars for metabolic flux analysis. Anal Biochem 425:183–188PubMedCrossRefGoogle Scholar
  3. 3.
    Blau K, Halket JM (1993) Handbook of derivatives for chromatography. Wiley, Chichester, UKGoogle Scholar
  4. 4.
    Villas-Bôas SG, Smart KF, Sivakumaran S, Lane GA (2011) Alkylation or silylation for analysis of amino and non-amino organic acids by GC-MS? Metabolites 1:3–20CrossRefGoogle Scholar
  5. 5.
    Villas-Bôas SG, Mas S, Akesson M, Smedsgaard J, Nielsen J (2005) Mass spectrometry in metabolome analysis. Mass Spectrom Rev 24:613–646PubMedCrossRefGoogle Scholar
  6. 6.
    Villas-Bôas SG, Koulman A, Lane GA (2007) Analytical methods from the perspective of method standardization. In: Jewett MC, Nielsen J (eds) Topics in current genetics - metabolomics. Elsevier-Verlag, Berlin, Germany, p 11CrossRefGoogle Scholar
  7. 7.
    Sparkman OD, Penton Z, Kitson FG (2011) Gas chromatography and mass spectrometry: a practical guide, 2nd edn. Academic, USA, pp 185–187Google Scholar
  8. 8.
    Thurman EM, Mills MS (1998) Solid phase extraction: principles and practice. Wiley Europe, Hoboken, USA, p 372Google Scholar
  9. 9.
    Little JL (1999) Artifacts in trimethylsilyl derivatization reactions and ways to avoid them. J Chromatogr A 844:1–22PubMedCrossRefGoogle Scholar
  10. 10.
    Lee WN, Byerley LO, Bergner EA, Edmond J (1991) Mass isotopomer analysis: theoretical and practical considerations. Biol Mass Spectrom 20:451–458PubMedCrossRefGoogle Scholar
  11. 11.
    Nanchen A, Fuhrer T, Sauer U (2007) Determination of metabolic flux ratios from 13C experiments and gas chromatography–mass spectrometry data: protocol and principles. In: Weckwerth W (ed) Metabolomics: methods and protocols. Humana, Totowa, NJ, pp 177–197Google Scholar
  12. 12.
    Böhlke JK, De Laeter JR, De Bièvre P et al (2005) Isotopic compositions of the elements, 2001. J Phys Chem Ref Data 34:57–67CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Mohamed Koubaa
    • 1
  • Brigitte Thomasset
    • 2
  • Albrecht Roscher
    • 3
  1. 1.Department of Molecular GeneticsThe Ohio State UniversityColumbusUSA
  2. 2.Universite de Technologie de Compiegne, CNRS-FRE 3580, Gènie Enzymatique et Cellulaire, Centre de Recherche de RoyallieuCompiègne CedexFrance
  3. 3.Génie Enzymatique et Cellulaire, FRE CNRS 3580Université de PicardieAmiens CedexFrance

Personalised recommendations