Fast sampling method for mammalian cell metabolic analyses using liquid chromatography-mass spectrometry
Fast sampling method for mammalian cell metabolic analyses using liquid chromatography-mass spectrometry
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DOI：
10.1038/nprot.2014.198
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发表时间：
2015-01-01
期刊：
影响因子：
14.8
通讯作者：
Vorholt, Julia A.
中科院分区：
文献类型：
--
作者：
Martano, Giuseppe;Delmotte, Nathanael;Vorholt, Julia A.
代谢组学已成为解决生物学问题的有力工具。液相色谱-质谱联用（LCLC-MS）广泛用于代谢表征，包括靶向和非靶向方法。尽管最近有创新，但这项技术的一个关键方面是用于准确数据分析的样品制备。在本方案中，我们提出了一种用于贴壁细胞培养物中哺乳动物细胞代谢分析的稳健且适应性强的工作流程，特别适用于通过LCLC-MS进行定性和定量中心代谢物表征。每个样品由在盖玻片上生长的600，000个哺乳动物细胞组成，允许快速完整地转移细胞用于代谢物提取或培养基交换，例如，用于标记实验。采样程序包括在水中液流中快速有效的清洗步骤，这可以减少交叉污染和基质效应，同时最大限度地减少对细胞代谢稳态的干扰;随后是淬灭细胞代谢。后者通过使用用甲酸酸化的-20 ° C冷甲醇乙腈混合物，然后冷冻干燥、代谢物提取和LCLC-MS来实现。该方案需要2秒用于细胞取样直到淬灭，并且当应用所提供的纳米级LCLC-MS方法时，整个方案每个样品总共需要1.5小时。
Metabolomics has emerged as a powerful tool for addressing biological questions. Liquid chromatography coupled with mass spectrometry (LCLC-MS) is widely used for metabolic characterization, including targeted and untargeted approaches. Despite recent innovations, a crucial aspect of this technique is the sample preparation for accurate data analyses. In this protocol, we present a robust and adaptable workflow for metabolic analyses of mammalian cells from adherent cell cultures, which is particularly suited for qualitative and quantitative central metabolite characterization by LCLC-MS. Each sample consists of 600,000 mammalian cells grown on cover glasses, allowing for fast and complete transfer of the cells for metabolite extraction or medium exchange, e.g., for labeling experiments. The sampling procedure includes a fast and efficient washing step in liquid flow in water, which reduces cross-contamination and matrix effects while minimizing perturbation of the metabolic steady state of the cells; it is followed by quenching cell metabolism. The latter is achieved by using a -20 degrees C cold methanol acetonitrile mixture acidified with formic acid, followed by freeze drying, metabolite extraction and LCLC-MS. The protocol requires 2 s for cell sampling until quenching, and the entire protocol takes a total of 1.5 h per sample when the provided nanoscale LCLC-MS method is applied.