Skip to main content
SpringerLink
Log in

Quantification of 13C Enrichments and Isotopomer Abundances for Metabolic Flux Analysis Using 1D NMR Spectroscopy

  • Protocol
  • First Online:
Plant Metabolic Flux Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1090))

Abstract

The analysis of stable isotope incorporation following feeding of 13C-labeled precursors to plant tissues provides the constraints necessary for metabolic flux analysis. This protocol describes the use of one-dimensional 1H and 13C nuclear magnetic resonance spectroscopy for the quantification of 13C enrichments and isotopomer abundances in mixtures of metabolites or hydrolyzed biomass components.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ratcliffe RG, Shachar-Hill Y (2006) Measuring multiple fluxes through plant metabolic networks. Plant J 45:490–511

    Article  PubMed  CAS  Google Scholar 

  2. O'Grady J, Schwender J, Shachar-Hill Y et al (2012) Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies. J Exp Bot 63:2293–2308

    Article  PubMed  Google Scholar 

  3. Allen DK, Ratcliffe RG (2009) Quantification of isotope label. In: Schwender J (ed) Plant Metabolic Networks. Springer, New York, pp 105–149

    Chapter  Google Scholar 

  4. Wiechert W (2001) 13C Metabolic Flux Analysis. Metab Eng 3:196–206

    Google Scholar 

  5. Wiechert W, Möllney M, Isermann N et al (1999) Bidirectional reaction steps in metabolic networks: III. Explicit solution and analysis of isotopomer labeling systems. Biotechnol Bioen 66:69–85

    Article  CAS  Google Scholar 

  6. Ratcliffe RG, Shachar-Hill Y (2005) Revealing metabolic phenotypes in plants: inputs from NMR analysis. Biol Rev Camb Philos Soc 80:27–43

    Article  PubMed  CAS  Google Scholar 

  7. Alonso AP, Goffman FD, Ohlrogge JB et al (2007) Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos. Plant J 52:296–308

    Article  PubMed  CAS  Google Scholar 

  8. Williams TC, Miguet L, Masakapalli SK et al (2008) Metabolic network fluxes in heterotrophic Arabidopsis cells: stability of the flux distribution under different oxygenation conditions. Plant Physiol 148:704–718

    Article  PubMed  CAS  Google Scholar 

  9. Masakapalli SK, Le Lay P, Huddleston JE et al (2010) Subcellular flux analysis of central metabolism in a heterotrophic Arabidopsis cell suspension using steady-state stable isotope labeling. Plant Physiol 152:602–619

    Article  PubMed  CAS  Google Scholar 

  10. Sriram G, Fulton DB, Iyer VV et al (2004) Quantification of compartmented metabolic fluxes in developing soybean embryos by employing biosynthetically directed fractional 13C labeling, two-dimensional [13C, 1H] nuclear magnetic resonance, and comprehensive isotopomer balancing. Plant Physiol 136:3043–3057

    Article  PubMed  CAS  Google Scholar 

  11. Ettenhuber C, Radykewicz T, Kofer W et al (2005) Metabolic flux analysis in complex isotopolog space. Recycling of glucose in tobacco plants. Phytochemistry 66:323–335

    Article  PubMed  CAS  Google Scholar 

  12. Ettenhuber C, Spielbauer G, Margl L et al (2005) Changes in flux pattern of the central carbohydrate metabolism during kernel development in maize. Phytochemistry 66:2632–2642

    Article  PubMed  CAS  Google Scholar 

  13. Sriram G, Fulton DB, Shanks JV (2007) Flux quantification in central carbon metabolism of Catharanthus roseus hairy roots by 13C labeling and comprehensive bondomer balancing. Phytochemistry 68:2243–2257

    Article  PubMed  CAS  Google Scholar 

  14. Giraudeau P, Massou S, Robin Y et al (2011) Ultrafast quantitative 2D NMR: an efficient tool for the measurement of specific isotopic enrichments in complex biological mixtures. Anal Chem 15:3112–3119

    Article  Google Scholar 

  15. Giraudeau P, Cahoreau E, Massou S et al (2012) UFJCOSY: A Fast 3D NMR Method for Measuring Isotopic Enrichments in Complex Samples. Chemphyschem 13:3098–3101

    Article  PubMed  CAS  Google Scholar 

  16. Massou S, Nicolas N, Letisse F et al (2007) Application of 2D-TOCSY NMR to the measurement of specific 13C-enrichments in complex mixtures of 13C-labeled metabolites. Metab Eng 9:252–257

    Article  PubMed  CAS  Google Scholar 

  17. Thrippleton MJ, Keeler J (2003) Elimination of Zero-Quantum interference in two-dimensional NMR spectra. Angew Chem Int Ed 42:3938–3941

    Article  CAS  Google Scholar 

  18. Peterson DJ, Loening NM (2007) QQ-HSQC: a quick, quantitative heteronuclear correlation experiment for NMR spectroscopy. Magn Reson Chem 45:937–941

    Article  PubMed  CAS  Google Scholar 

  19. Cahoreau E, Peyriga L, Hubert J et al (2012) Isotopic profiling of 13C-labeled biological samples by two-dimensional heteronuclear J-resolved nuclear magnetic resonance spectroscopy. Anal Biochem 427:158–163

    Article  PubMed  CAS  Google Scholar 

  20. Ludwig C, Viant MR (2010) Two-dimensional J-resolved NMR spectroscopy: review of a key methodology in the metabolomics toolbox. Phytochem Anal 21:22–32

    Article  PubMed  CAS  Google Scholar 

  21. Libourel IGL, Gehan JP, Shachar-Hill Y (2007) Design of substrate label for steady state flux measurements in plant systems using the metabolic network of Brassica napus embryos. Phytochemistry 68:2211–2221

    Article  PubMed  CAS  Google Scholar 

  22. Rontein D, Dieuaide-Noubhani M, Dufoure EJ et al (2002) The metabolic architecture of plant cells: stability of central metabolism and flexibility of anabolic pathways during the growth cycle of tomato cells. J Biochem 46:43948–43960

    Google Scholar 

  23. Allen DK, Shachar-Hill Y, Ohlrogge JB (2007) Compartment-specific labeling information in 13C metabolic flux analysis of plants. Phytochemistry 68:2197–2210

    Article  PubMed  CAS  Google Scholar 

  24. Chen X, Alonso AP, Allen DK et al (2011) Synergy between 13C-metabolic flux analysis and flux balance analysis for understanding metabolic adaptation to anaerobiosis in E. coli. Metab Eng 13:38–48

    Article  PubMed  Google Scholar 

  25. Kruger NJ, Troncoso-Ponce MA, Ratcliffe RG (2008) 1H NMR of metabolite fingerprinting and metabolomic analysis of perchloric acid extracts from plant tissues. Nat Protocols 3:1001–1012

    Article  CAS  Google Scholar 

  26. Wiechert W, Möllney M, Petersen S et al (2001) A universal framework for 13C metabolic flux analysis. Metab Eng 3:265–283

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

TCRW was supported by a Petrobras (Petroleo Brasileiro S.A.) postdoctoral grant. SKM acknowledges financial support from the University of Oxford (Clarendon Scholarship), Exeter College (Mr Krishna Pathak Scholarship), and a UK Overseas Research Student award.

Author information

Authors and Affiliations

  1. Department of Plant Sciences, University of Oxford, Oxford, UK

    Shyam K. Masakapalli & R. George Ratcliffe

  2. Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil

    Thomas C. R. Williams

Authors
  1. Shyam K. Masakapalli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. George Ratcliffe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas C. R. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. INRA Bordeaux Université Bordeaux Segalen, Villenave d'Ornon, France

    Martine Dieuaide-Noubhani

  2. Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA

    Ana Paula Alonso

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Masakapalli, S.K., Ratcliffe, R.G., Williams, T.C.R. (2014). Quantification of 13C Enrichments and Isotopomer Abundances for Metabolic Flux Analysis Using 1D NMR Spectroscopy. In: Dieuaide-Noubhani, M., Alonso, A. (eds) Plant Metabolic Flux Analysis. Methods in Molecular Biology, vol 1090. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-688-7_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-688-7_5

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-687-0

  • Online ISBN: 978-1-62703-688-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation