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

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

Research output: Contribution to journalArticlepeer-review

252 Scopus citations

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 13C-labeled carbon sources, with detection of 13C-assimilation by liquid chromatography-tandem MS. It enables absolute quantitation of several dozen metabolites over ∼1 week of work.

Original languageEnglish (US)
Pages (from-to)1299-1311
Number of pages13
JournalNature Protocols
Volume3
Issue number8
DOIs
StatePublished - 2008

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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