Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach View Full Text


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Article Info

DATE

2008-08

AUTHORS

Bryson D Bennett, Jie Yuan, Elizabeth H Kimball, Joshua D Rabinowitz

ABSTRACT

This protocol provides a method for quantitating the intracellular concentrations of endogenous metabolites in cultured cells. The cells are grown in stable isotope-labeled media to near-complete isotopic enrichment and then extracted in organic solvent containing unlabeled internal standards in known concentrations. The ratio of endogenous metabolite to internal standard in the extract is determined using mass spectrometry (MS). The product of this ratio and the unlabeled standard amount equals the amount of endogenous metabolite present in the cells. The cellular concentration of the metabolite can then be calculated on the basis of intracellular volume of the extracted cells. The protocol is exemplified using Escherichia coli and primary human fibroblasts fed uniformly with (13)C-labeled carbon sources, with detection of (13)C-assimilation by liquid chromatography-tandem MS. It enables absolute quantitation of several dozen metabolites over approximately 1 week of work. More... »

PAGES

1299-1311

References to SciGraph publications

  • 2006-10. Kinetic flux profiling of nitrogen assimilation in Escherichia coli in NATURE CHEMICAL BIOLOGY
  • 2006-01. A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites in JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
  • 2007-05. Isotope ratio-based profiling of microbial folates in JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
  • Identifiers

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    http://scigraph.springernature.com/pub.10.1038/nprot.2008.107

    DOI

    http://dx.doi.org/10.1038/nprot.2008.107

    DIMENSIONS

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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/18714298


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