Tissue disruption and extraction methods for metabolic profiling of an invertebrate sentinel species
Tissue disruption and extraction methods for metabolic profiling of an invertebrate sentinel species
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DOI：
10.1007/s11306-011-0377-1
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发表时间：
2012-10-01
期刊：
影响因子：
3.6
通讯作者：
Bundy, Jacob G.
中科院分区：
文献类型：
--
作者：
Liebeke, Manuel;Bundy, Jacob G.
组织的代谢分析需要特别注意，因为样品均质化将消除细胞成分的划分。这种分配的损失导致提取物中的蛋白质和代谢物不稳定，因此代谢物提取方案需要确保大分子非常快速地失活以及代谢物的溶解。组织代谢组分析有许多已发表的方法，但没有普遍接受的标准，并且缺乏可测量的质量基准。我们开发了一种方案，用于在先前的生物学知识和基于数据的指标的指导下，对蚯蚓 Lumbricus rubellus 进行有效的组织破坏和代谢物提取。特别是，我们发现了红乳杆菌组织提取物异常程度的不稳定性，并评估了不同的方法（例如加热和过滤）来抵消这种不稳定性。最后，我们使用三个分析平台（H-1 NMR 光谱、GC-MS 和直接输注 FT-ICR-MS）评估了四种不同的溶剂系统用于综合代谢物提取，并比较了用于组织破坏的珠打和低温研磨。最初，我们根据常见的分析标准（例如检测到的峰的数量和总强度）对方法进行排名，以便比较方案。由于样品不稳定，这些评估方案适用性的方法被证明不足以判断蚯蚓组织提取方法。不应假定现有的组织提取方案自动适用于新物种。
Metabolic profiling of tissues needs special attention, because the compartmentalization of cellular constituents will be abolished by sample homogenization. This loss of partitioning leads to protein and metabolite instability in extracts, and therefore metabolite extraction protocols need to ensure very rapid inactivation of macromolecules as well as solubilization of metabolites. There are many published methods for tissue metabolome analysis, but no universally accepted standard, and a lack of measurable quality benchmarks. We developed a protocol for efficient tissue disruption and metabolite extraction of the earthworm Lumbricus rubellus guided by prior biological knowledge as well as metrics based on the data. In particular, we identified an unusual degree of instability of L. rubellus tissue extracts, and evaluated different approaches such as heating and filtration to counteract this. Finally, we evaluated four different solvent systems for comprehensive metabolite extraction using three analytical platforms (H-1 NMR spectroscopy, GC-MS, and direct-infusion FT-ICR-MS), and also compared bead-beating and cryogenic milling for tissue disruption. Initially we ranked methods by common analytical criteria (e.g. numbers and total intensity of detected peaks) in order to compare protocols. These approaches to assess protocol suitability proved to be inadequate to judge earthworm tissue extraction methods because of sample instability. Existing tissue extraction protocols should not be assumed to be automatically applicable to novel species.