Quantitative Evaluation of Intracellular Metabolite Extraction Techniques for Yeast Metabolomics
Quantitative Evaluation of Intracellular Metabolite Extraction Techniques for Yeast Metabolomics
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DOI：
10.1021/ac900999t
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发表时间：
2009-09-01
影响因子：
7.4
通讯作者：
Heijnen, Joseph J.
中科院分区：
文献类型：
--
作者：
Canelas, Andre B.;ten Pierick, Angela;Heijnen, Joseph J.
准确测定细胞内代谢物水平需要经过充分验证的采样和样品处理程序。存在几种代谢物提取方法，但文献中关于每种技术的充分性和性能存在矛盾。采用严格的定量方法，我们重新评估了5种方法（热水，HW;沸腾乙醇，BE;氯仿甲醇，CM;甲醇中冻融，FTM;酸性乙腈-甲醇，AANM）用于从S.酿酒酵母细胞研究了两种培养模式（分批和恒化器），以检查生长条件依赖性，并采用了三个目标平台（两个LC-MS和一个GC/MS），以排除分析偏倚。此外，对于代谢物回收率的测定，我们应用了一种基于在样品处理的不同阶段加入（13）C-标记内标物的新方法。我们发现，提取方法的选择可以极大地影响测得的代谢物水平，在某种程度上，对于某些代谢物，甚至可以逆转生长条件之间的变化方向。在有效性和代谢物回收率方面，BE和CM达到了最佳性能，其分析的代谢物水平几乎相同。根据我们的结果，AANM在酵母中的表现不佳，FTM不能被认为是一种适当的提取方法，因为它不能确保酶活性的灭活。
Accurate determination of intracellular metabolite levels requires well-validated procedures for sampling and sample treatment. Several methods exist for metabolite extraction, but the literature is contradictory regarding the adequacy and performance of each technique. Using a strictly quantitative approach, we have re-evaluated five methods (hot water, HW; boiling ethanol, BE; chloroform methanol, CM; freezing-thawing in methanol, FTM; acidic acetonitrile-methanol, AANM) for the extraction of 44 intracellular metabolites (phosphorylated intermediates, amino acids, organic acids, nucleotides) from S. cerevisiae cells. Two culture modes were investigated (batch and chemostat) to check for growth condition dependency, and three targeted platforms were employed (two LC-MS and one GC/MS) to exclude analytical bias. Additionally, for the determination of metabolite recoveries, we applied a novel approach based on addition of (13)C-labeled internal standards at different stages of sample processing. We found that the choice of extraction method can drastically affect measured metabolite levels, to an extent that for some metabolites even the direction of changes between growth conditions can be inverted. The best performances, in terms of efficacy and metabolite recoveries, were achieved with BE and CM, which yielded nearly identical levels for the metabolites analyzed. According to our results, AANM performs poorly in yeast and FTM cannot be considered adequate as an extraction method, as it does not ensure inactivation of enzymatic activity.