Identifying decomposition products in extracts of cellular metabolites.
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.
Kimball
Elizabeth
E
Department of Chemistry and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08542, USA.
Rabinowitz
Joshua D
JD
D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000378:metabolism*; D013058:Mass Spectrometry; D000432:Methanol / Q000737:chemistry; D012015:Reference Standards; D014867:Water / Q000737:chemistry
D014867:Water; D000432:Methanol