Strategy for choosing extraction procedures for NMR-based metabolomic analysis of mammalian cells
Strategy for choosing extraction procedures for NMR-based metabolomic analysis of mammalian cells
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DOI：
10.1007/s00216-011-5310-y
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发表时间：
2011-10-01
影响因子：
4.3
通讯作者：
Akoka, Serge
中科院分区：
文献类型：
--
作者：
Martineau, Estelle;Tea, Illa;Akoka, Serge
哺乳动物细胞的代谢组学分析可以应用于包括医学和毒理学在内的多个领域。它需要获得可重复的、稳健的、可靠的和同质的生物数据集。必须特别注意提取程序的效率和可靠性。尽管最近的一些研究已经涉及优化特定基质和分析技术的特定方案，但没有通用的方法来检测整个细胞代谢组。在这里，我们提出了一个策略，选择提取程序从贴壁哺乳动物细胞的代谢组的全球NMR分析。细胞淬灭后，使用以下不同极性的溶剂系统之一从细胞中提取细胞内代谢物：高氯酸、乙腈/水、甲醇、甲醇/水和甲醇/氯仿/水。使用H-1核磁共振（NMR）光谱分析亲水性代谢产物谱。我们提出了一个原始的几何表示的代谢产物，反映了提取方法的效率。在哺乳动物细胞的基于NMR的分析的情况下，该方法证明了通过使用甲醇或甲醇/氯仿/水提取更高部分的细胞内代谢物。优选的方法进行评估的生物变异性研究代谢变化引起的表型的四种不同的人乳腺癌细胞系，表明所选择的提取程序是一个有前途的工具，代谢组学和代谢组学研究的哺乳动物细胞。本文提出的比较提取方法的策略适用于各种系统的基于NMR的代谢组学研究。
Metabolomic analysis of mammalian cells can be applied across multiple fields including medicine and toxicology. It requires the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Particular attention must be paid to the efficiency and reliability of the extraction procedure. Even though a number of recent studies have dealt with optimizing a particular protocol for specific matrices and analytical techniques, there is no universal method to allow the detection of the entire cellular metabolome. Here, we present a strategy for choosing extraction procedures from adherent mammalian cells for the global NMR analysis of the metabolome. After the quenching of cells, intracellular metabolites are extracted from the cells using one of the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol, methanol/water, and methanol/chloroform/water. The hydrophilic metabolite profiles are analysed using H-1 nuclear magnetic resonance (NMR) spectroscopy. We propose an original geometric representation of metabolites reflecting the efficiency of extraction methods. In the case of NMR-based analysis of mammalian cells, this methodology demonstrates that a higher portion of intracellular metabolites are extracted by using methanol or methanol/chloroform/water. The preferred method is evaluated in terms of biological variability for studying metabolic changes caused by the phenotype of four different human breast cancer cell lines, showing that the selected extraction procedure is a promising tool for metabolomic and metabonomic studies of mammalian cells. The strategy proposed in this paper to compare extraction procedures is applicable to NMR-based metabolomic studies of various systems.