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...

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Bibliographic Details
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
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2006.07.038