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 language | English (US) |
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Pages (from-to) | 273-280 |
Number of pages | 8 |
Journal | Analytical Biochemistry |
Volume | 358 |
Issue number | 2 |
DOIs | |
State | Published - Nov 15 2006 |
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Biophysics
- Biochemistry
- Cell Biology
Keywords
- Bacteria
- Extraction
- LC-MS/MS
- Metabolism
- Metabolomics
- Sampling
- Small molecule
- Stability
- Triple quadrupole