Evaluation and Optimization of Metabolome Sample Preparation Methods for Saccharomyces cerevisiae
Evaluation and Optimization of Metabolome Sample Preparation Methods for Saccharomyces cerevisiae
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DOI：
10.1021/ac302881e
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发表时间：
2013-02-19
影响因子：
7.4
通讯作者：
Kim, Kyoung Heon
中科院分区：
文献类型：
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作者：
Kim, Sooah;Lee, Do Yup;Kim, Kyoung Heon
代谢组采样是决定代谢组学数据质量的重要因素之一。代谢物样品制备的主要步骤包括猝灭和代谢物提取。酿酒酵母最常用的方法是在-40℃下用60%(v/v)冷甲醇猝灭，最近将这种方法改进为用纯甲醇在-40℃下无泄漏冷甲醇猝灭，沸腾乙醇(75%，v/v)和冷纯甲醇是酿酒酵母最常用的提取溶剂。在本研究中，利用气相色谱/飞行时间质谱仪分析了110种已鉴定的酿酒酵母细胞内代谢物，对包括上述方法在内的代谢组采样方案进行了评估。根据我们的结果，快速过滤后加适量的水冲洗可以最大限度地减少细胞渗漏造成的代谢物损失和胞外代谢物的污染，对于代谢物的提取，乙腈/水(1：1，v/v)在-20℃下是最有效的。这些结果表明，对现有方法的系统评价和针对每种微生物的定制方法的开发对于代谢组样品的制备至关重要，以促进代谢组可靠和准确的分析。
Metabolome sampling is one of the most important factors that determine the quality of metabolomics data. The main steps in metabolite sample preparation include quenching and metabolite extraction. Quenching with 60% (v/v) cold methanol at -40 degrees C has been most commonly used for Saccharomyces cerevisiae, and this method was recently modified as "leakage-free cold methanol quenching" using pure methanol at -40 degrees C. Boiling ethanol (75%, v/v) and cold pure methanol are the most widely used extraction solvents for S. cerevisiae. In the present study, metabolome sampling protocols, including the above methods, were evaluated by analyzing 110 identified intracellular metabolites of S. cerevisiae using gas chromatography/time-of-flight mass spectrometry. According to our results, fast filtration followed by washing with an appropriate volume of water can minimize the metabolite loss due to cell leakage as well as the contamination by extracellular metabolites, For metabolite extraction, acetonitrile/water mixture (1:1, v/v) at -20 degrees C was the most effective. These results imply that the systematic evaluation of existing methods and the development of customized methods for each microorganism are critical for metabolome sample preparation to facilitate the reliable and accurate analysis of metabolome.