Identifying decomposition products in extracts of cellular metabolites

Analytical Biochemistry
Elizabeth Kimball, Joshua D Rabinowitz

Abstract

Most methods of analyzing intracellular metabolites require extraction of metabolites from the cells. A concern in these methods is underestimation of metabolite levels due to incomplete extraction. In comparing extraction methods, then, it would seem that the best method for extracting a particular metabolite is the one that gives the largest yield. In extracting Escherichia coli with different methanol:water mixtures, we observed that >or=50% water gave an increased yield of nucleosides and bases compared with <or=20% water, as determined by liquid chromatography-tandem mass spectrometry analysis of the resulting extracts. Spiking of the extracts with isotope-labeled nucleotides revealed, however, that the high yield of nucleosides and bases occurred due to decomposition of nucleotides in the water-rich condition, not due to good extraction. Spiking combined with isotope labeling provides a general approach to detecting decomposition products in extracts of cellular metabolites. For extraction of E. coli with methanol:water, cold temperature and a high methanol fraction minimize artifacts due to metabolite decomposition.

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Citations

Apr 11, 2008·Analytical Chemistry·Qihui NiRobert T Kennedy
Jul 10, 2007·Journal of the American Chemical Society·Jie Yuan, Joshua D Rabinowitz
Jun 30, 2009·Nature Chemical Biology·Bryson D BennettJoshua D Rabinowitz
Aug 21, 2008·Nature Protocols·Bryson D BennettJoshua D Rabinowitz
Aug 21, 2008·Nature Protocols·Jie YuanJoshua D Rabinowitz
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