Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach.
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.
Bennett
Bryson D
BD
Department of Chemistry and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, USA.
Yuan
Jie
J
Kimball
Elizabeth H
EH
Rabinowitz
Joshua D
JD
D000818:Animals; D002244:Carbon / Q000378:metabolism*; D002247:Carbon Isotopes; D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D003470:Culture Media; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D008322:Mammals / Q000378:metabolism; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
D002247:Carbon Isotopes; D003470:Culture Media; D002244:Carbon