Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach
Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach
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DOI：
10.1038/nprot.2008.107
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Rabinowitz, Joshua D.
中科院分区：
文献类型：
--
作者：
Bennett, Bryson D.;Yuan, Jie;Rabinowitz, Joshua D.
该方案提供了一种定量培养细胞中内源性代谢产物细胞内浓度的方法。这些细胞在稳定的同位素标记的培养基中生长至接近完整的同位素富集，然后在包含已知浓度的未标记内标准的有机溶剂中提取。使用质谱法（MS）确定内源代谢物与提取物中的内标的比率。该比率和未标记的标准量的乘积等于细胞中存在的内源代谢物的量。然后可以根据提取的细胞的细胞内体积来计算代谢物的细胞浓度。该方案使用大肠杆菌和原代人成纤维细胞均匀地用C-13标记的碳源呈现，并通过液相色谱倾斜MS检测到C-13-鉴别。它可以在类似于1周的工作中绝对定量数十几十个代谢产物。
This protocol provides a method for quantitating the intracellular concentrations of endogenous metabolites in cultured cells. The cells are grown in stable isotope-labeled media to near-complete isotopic enrichment and then extracted in organic solvent containing unlabeled internal standards in known concentrations. The ratio of endogenous metabolite to internal standard in the extract is determined using mass spectrometry (MS). The product of this ratio and the unlabeled standard amount equals the amount of endogenous metabolite present in the cells. The cellular concentration of the metabolite can then be calculated on the basis of intracellular volume of the extracted cells. The protocol is exemplified using Escherichia coli and primary human fibroblasts fed uniformly with C-13-labeled carbon sources, with detection of C-13-assimilation by liquid chromatography-tandem MS. It enables absolute quantitation of several dozen metabolites over similar to 1 week of work.