Department of Parasitology, Unit of Molecular Parasitology, Institute of Tropical Medicine, 2000 Antwerp, Belgium.
Anal Bioanal Chem. 2010 Nov;398(5):2059-69. doi: 10.1007/s00216-010-4139-0. Epub 2010 Sep 8.
Comparative metabolomics of Leishmania species requires the simultaneous identification and quantification of a large number of intracellular metabolites. Here, we describe the optimisation of a comprehensive metabolite extraction protocol for Leishmania parasites and the subsequent optimisation of the analytical approach, consisting of hydrophilic interaction liquid chromatography coupled to LTQ-orbitrap mass spectrometry. The final optimised protocol starts with a rapid quenching of parasite cells to 0 °C, followed by a triplicate washing step in phosphate-buffered saline. The intracellular metabolome of 4 × 10(7) parasites is then extracted in cold chloroform/methanol/water 20/60/20 (v/v/v) for 1 h at 4 °C, resulting in both cell disruption and comprehensive metabolite dissolution. Our developed metabolomics platform can detect approximately 20% of the predicted Leishmania metabolome in a single experiment in positive and negative ionisation mode.
要对利什曼原虫物种进行比较代谢组学研究，需要同时鉴定和定量大量的细胞内代谢物。在这里，我们描述了一种综合代谢物提取方案的优化，该方案适用于利什曼原虫寄生虫，随后优化了分析方法，包括亲水相互作用液相色谱与 LTQ-orbitrap 质谱联用。最终优化的方案从将寄生虫细胞快速淬灭到 0°C 开始，随后在磷酸盐缓冲盐水（phosphate-buffered saline）中重复洗涤 3 次。然后在 4°C 下，用冷的氯仿/甲醇/水（20/60/20，v/v/v）将 4×10(7)个寄生虫的细胞内代谢组提取 1 小时，从而实现细胞破裂和全面代谢物溶解。我们开发的代谢组学平台可以在正离子和负离子模式下单次实验中检测到约 20%的预测利什曼原虫代谢组。
