Cross-Platform Comparison of Methods for Quantitative Metabolomics of Primary Metabolism
Cross-Platform Comparison of Methods for Quantitative Metabolomics of Primary Metabolism
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DOI：
10.1021/ac8022857
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发表时间：
2009-03-15
影响因子：
7.4
通讯作者：
Zamboni, Nicola
中科院分区：
文献类型：
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作者：
Buescher, Joerg Martin;Czernik, Dominika;Zamboni, Nicola
定量代谢组学正处于紧张的发展中，尚未出现普遍接受的标准分析技术。所采用的分析方法大多是根据有根据的猜测选择的。到目前为止，还没有系统的跨平台比较不同的分离和检测方法的定量代谢组学。一般来说，代谢物的色谱分离，然后在质谱仪（MS）中对其进行选择性检测，在灵敏度和分离能力方面是最有前途的方法。使用91种代谢物（包括糖酵解、戊糖磷酸途径、三羧酸（TCA）循环、氧化还原代谢、氨基酸和核苷酸）的确定混合物，我们比较了设计用于分析这些主要极性初级代谢物的六种分离方法，气相色谱（GC）、液相色谱（LC）和毛细管电泳（CE）各两种方法。对于单一平台上的分析，LC提供了通用性和稳健性的最佳组合。如果可以使用第二个平台，最好用GC作为补充。只有液相分离系统才能处理大的极性代谢物，如含有多个磷酸基团的代谢物。如通过用C-13标记的酵母提取物补充定义的混合物所评估的，基质效应是所有平台上的常见现象。因此，合适的内标物，如C-13标记的生物质提取物，是定量代谢组学与任何方法的强制性。
Quantitative metabolomics is under intense development, and no commonly accepted standard analytical technique has emerged, yet. The employed analytical methods were mostly chosen based on educated guesses. So far, there has been no systematic cross-platform comparison of different separation and detection methods for quantitative metabolomics. Generally, the chromatographic separation of metabolites followed by their selective detection in a mass spectrometer (MS) is the most promising approach in terms of sensitivity and separation power. Using a defined mixture of 91 metabolites (coveting glycolysis, pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, redox metabolism, amino acids, and nucleotides), we compared six separation methods designed for the analysis of these mostly very polar primary metabolites, two methods each for gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). For analyses on a single platform, LC provides the best combination of both versatility and robustness. If a second platform can be used, it is best complemented by GC. Only liquid-phase separation systems can handle large polar metabolites, such as those containing multiple phosphate groups. As assessed by supplementing the defined mixture with C-13-labeled yeast extracts, matrix effects are a common phenomenon on all platforms. Therefore, suitable internal standards, such as C-13-labeled biomass extracts, are mandatory for quantitative metabolomics with any methods.