Simultaneous quantification of energetically important metabolites in various cell types by CZE
Language English Country Germany Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- Keywords
- Metabolomics, Nucleotides, Paracoccus denitrificans, Stem cells, System biology,
- MeSH
- Acetyl Coenzyme A analysis MeSH
- Adenosine Triphosphate analysis MeSH
- Chemistry Techniques, Analytical methods standards MeSH
- Cytidine Triphosphate analysis MeSH
- Embryonic Stem Cells chemistry MeSH
- Guanosine Triphosphate analysis MeSH
- Humans MeSH
- Limit of Detection MeSH
- Paracoccus denitrificans chemistry MeSH
- Reproducibility of Results MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Acetyl Coenzyme A MeSH
- Adenosine Triphosphate MeSH
- Cytidine Triphosphate MeSH
- Guanosine Triphosphate MeSH
A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field-enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on-line pre-concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.
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