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

Nature Protocols volume 3, pages 1299–1311 (2008)Cite this article

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.

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Figure 1
Figure 2
Figure 3: Schematic of potential mass spectrometry peak intensities obtained via Steps 2–6 for a 4 carbon compound after 13C labeling.
Figure 4: Creating filter culture from batch culture (Step 2A(vi)).
Figure 5
Figure 6: Quenching and extraction of a filter culture (Step 2A(x)).
Figure 7
Figure 8: Representative extracted ion chromatograms of UTP.

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Acknowledgements

This research was supported by the Beckman Foundation, National Science Foundation–Dynamic Data-Driven Application Systems grant CNS-0540181, American Heart Association grant 0635188N, National Science Foundation CAREER award MCB-0643859, National Institutes of Health grant AI078063 and National Institutes of Health grant GM071508 for the Center of Quantitative Biology at Princeton University. We thank Wenyun Lu for contributing LC-MS expertise.

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Authors and Affiliations

  1. Department of Chemistry and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, 08544, New Jersey, USA

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

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Correspondence to Joshua D Rabinowitz.

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Bennett, B., Yuan, J., Kimball, E. et al. Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach. Nat Protoc 3, 1299–1311 (2008). https://doi.org/10.1038/nprot.2008.107

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