Identifying decomposition products in extracts of cellular metabolites

Elizabeth Kimball, Joshua D. Rabinowitz

Research output: Contribution to journalArticle

65 Scopus citations

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 ≥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.

Original languageEnglish (US)
Pages (from-to)273-280
Number of pages8
JournalAnalytical Biochemistry
Volume358
Issue number2
DOIs
StatePublished - Nov 15 2006

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Keywords

  • Bacteria
  • Extraction
  • LC-MS/MS
  • Metabolism
  • Metabolomics
  • Sampling
  • Small molecule
  • Stability
  • Triple quadrupole

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