GC-MS profiling of leukemia cells: an optimized preparation protocol for the intracellular metabolome†,Analytical Methods

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GC-MS profiling of leukemia cells: an optimized preparation protocol for the intracellular metabolome†
Analytical Methods ( IF 2.7 ) Pub Date : 2018-02-01 00:00:00 , DOI: 10.1039/c7ay02578e
Y. He _{1,

2,

3,

4} , Z. M. Zhang _{1,

2,

3,

4} , P. Ma _{1,

2,

3,

4} , H. C. Ji _{1,

2,

3,

4} , H. M. Lu _{1,

2,

3,

4}

Affiliation

Sample preparation is an important step in the untargeted cell metabolomics workflow. In this study, a gas chromatography-mass spectrometry (GC-MS)-based metabolome preparation protocol was optimized with the aim of achieving global, reliable and reproducible intracellular metabolite profiling of leukemia cells. We have evaluated and compared 8 sample preparation methods: 4 extraction solvents (i.e., pure methanol, aqueous acetonitrile, methanol/methanol/water and aqueous methanol) coupled with 2 common trimethylsilylation reagents (i.e., N,O-bis(trimethylsilyl)trifluoroacetamide and N-methyl-N-(trimethylsilyl)trifluoroacetamide). The extraction solvents and derivatization agents were evaluated for their suitability for leukemia cells using criteria based on the number of total detected metabolites, extraction efficiency and repeatability. Based on the overall performance, methanol/methanol/water was chosen as the optimal extraction solvent for covering the highest number of intracellular metabolites with the best reproducibility. The derivatization agent N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) proved to be more suitable for profiling of the leukemia intracellular metabolome with better repeatability than N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). This optimized method provides a robust, efficient and reproducible way to gather information on the metabolome of a leukemia cell. It also offers a solid basis to further understand the physiology of leukemia cells and unravel the mechanism of leukemia’s multidrug resistance from the perspective of metabolomics.

中文翻译：

白血病细胞的GC-MS分析：细胞内代谢组的优化制备方案†

样品制备是非靶向细胞代谢组学工作流程中的重要步骤。在这项研究中，基于气相色谱-质谱（GC-MS）的代谢物制备方案进行了优化，目的是实现白血病细胞的整体，可靠和可重现的细胞内代谢物谱分析。我们已经评价和比较8种样品制备方法：4的萃取溶剂（即，加上2种共同三甲基硅烷化试剂（纯甲醇，含水乙腈，甲醇/甲醇/水和含水甲醇）即，Ñ，ö双（三甲基硅烷基）三氟乙酰胺和ñ甲基ñ-（三甲基甲硅烷基）三氟乙酰胺）。基于总检测代谢物的数量，提取效率和可重复性，使用标准评估提取溶剂和衍生剂对白血病细胞的适用性。基于整体性能，选择甲醇/甲醇/水作为覆盖最多细胞内代谢物和最佳重现性的最佳提取溶剂。事实证明，衍生试剂N，O-双（三甲基甲硅烷基）三氟乙酰胺（BSTFA）比N-甲基-N更适合于白血病细胞内代谢组的分析-（三甲基甲硅烷基）三氟乙酰胺（MSTFA）。这种优化的方法为收集有关白血病细胞代谢组信息的方法提供了一种可靠，有效且可重现的方法。它还为从代谢组学的角度进一步了解白血病细胞的生理机制和阐明白血病对多种药物的耐药性提供了坚实的基础。

更新日期：2018-02-01

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