Identifying decomposition products in extracts of cellular metabolites

• Published in: Analytical biochemistry 2006-11, Vol.358 (2), p.273-280
• Main Authors: Kimball, Elizabeth, Rabinowitz, Joshua D.
• Format: Article
• Language: English
• Subjects: Bacteria; Chromatography, Liquid; Escherichia coli; Escherichia coli - metabolism; Extraction; LC–MS/MS; Mass Spectrometry; Metabolism; Metabolomics; Methanol - chemistry; Reference Standards; Sampling; Small molecule; Stability; Triple quadrupole; Water - chemistry
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2006.07.038

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 ⩾50% water gave an increased yield of nucleosides and bases compared with ⩽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.