Towards quantitative metabolomics of mammalian cells: Development of a metabolite extraction protocol
Metabolomics aims to quantify all metabolites within an organism, thereby providing valuable insight into the metabolism of cells. To study intracellular metabolites, they are first extracted from the cells. The ideal extraction procedure should immediately quench metabolism and quantitatively extra...
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| Published in | Analytical biochemistry Vol. 404; no. 2; pp. 155 - 164 |
|---|---|
| Main Authors | , , , , |
| Format | Journal Article |
| Language | English |
| Published |
United States
Elsevier Inc
15.09.2010
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| Subjects | |
| Online Access | Get full text |
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| Abstract | Metabolomics aims to quantify all metabolites within an organism, thereby providing valuable insight into the metabolism of cells. To study intracellular metabolites, they are first extracted from the cells. The ideal extraction procedure should immediately quench metabolism and quantitatively extract all metabolites, a significant challenge given the rapid turnover and physicochemical diversity of intracellular metabolites. We have evaluated several quenching and extraction solutions for their suitability for mammalian cells grown in suspension. Quenching with 60% methanol (buffered or unbuffered) resulted in leakage of intracellular metabolites from the cells. In contrast, quenching with cold isotonic saline (0.9% [w/v] NaCl, 0.5
°C) did not damage cells and effectively halted conversion of ATP to ADP and AMP, indicative of metabolic arrest. Of the 12 different extraction methods tested, cold extraction in 50% aqueous acetonitrile was superior to other methods. The recovery of a mixture of standards was excellent, and the concentration of extracted intracellular metabolites was higher than for the other methods tested. The final protocol is easy to implement and can be used to study the intracellular metabolomes of mammalian cells. |
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| AbstractList | Metabolomics aims to quantify all metabolites within an organism, thereby providing valuable insight into the metabolism of cells. To study intracellular metabolites, they are first extracted from the cells. The ideal extraction procedure should immediately quench metabolism and quantitatively extract all metabolites, a significant challenge given the rapid turnover and physicochemical diversity of intracellular metabolites. We have evaluated several quenching and extraction solutions for their suitability for mammalian cells grown in suspension. Quenching with 60% methanol (buffered or unbuffered) resulted in leakage of intracellular metabolites from the cells. In contrast, quenching with cold isotonic saline (0.9% [w/v] NaCl, 0.5 degrees C) did not damage cells and effectively halted conversion of ATP to ADP and AMP, indicative of metabolic arrest. Of the 12 different extraction methods tested, cold extraction in 50% aqueous acetonitrile was superior to other methods. The recovery of a mixture of standards was excellent, and the concentration of extracted intracellular metabolites was higher than for the other methods tested. The final protocol is easy to implement and can be used to study the intracellular metabolomes of mammalian cells. Metabolomics aims to quantify all metabolites within an organism, thereby providing valuable insight into the metabolism of cells. To study intracellular metabolites, they are first extracted from the cells. The ideal extraction procedure should immediately quench metabolism and quantitatively extract all metabolites, a significant challenge given the rapid turnover and physicochemical diversity of intracellular metabolites. We have evaluated several quenching and extraction solutions for their suitability for mammalian cells grown in suspension. Quenching with 60% methanol (buffered or unbuffered) resulted in leakage of intracellular metabolites from the cells. In contrast, quenching with cold isotonic saline (0.9% [w/v] NaCl, 0.5 °C) did not damage cells and effectively halted conversion of ATP to ADP and AMP, indicative of metabolic arrest. Of the 12 different extraction methods tested, cold extraction in 50% aqueous acetonitrile was superior to other methods. The recovery of a mixture of standards was excellent, and the concentration of extracted intracellular metabolites was higher than for the other methods tested. The final protocol is easy to implement and can be used to study the intracellular metabolomes of mammalian cells. |
| Author | Krömer, Jens O. Dietmair, Stefanie Timmins, Nicholas E. Gray, Peter P. Nielsen, Lars K. |
| Author_xml | – sequence: 1 givenname: Stefanie surname: Dietmair fullname: Dietmair, Stefanie email: s.dietmair@uq.edu.au – sequence: 2 givenname: Nicholas E. surname: Timmins fullname: Timmins, Nicholas E. – sequence: 3 givenname: Peter P. surname: Gray fullname: Gray, Peter P. – sequence: 4 givenname: Lars K. surname: Nielsen fullname: Nielsen, Lars K. – sequence: 5 givenname: Jens O. surname: Krömer fullname: Krömer, Jens O. |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/20435011$$D View this record in MEDLINE/PubMed |
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| SubjectTerms | Acetonitriles - chemistry Animals CHO Cells Chromatography, High Pressure Liquid Cricetinae Cricetulus Extraction Mammalian cell culture Metabolome Metabolomics Metabolomics - methods Methanol - chemistry Quenching Sodium Chloride - chemistry |
| Title | Towards quantitative metabolomics of mammalian cells: Development of a metabolite extraction protocol |
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