背景:样品处理策略的选择是代谢组学工作流程中的关键步骤。固相微萃取(SPME)是一种样品处理方法,具有在组织样品的非靶向代谢组学中使用的巨大潜力。然而,它的使用不像其他涉及组织收集步骤的标准协议那样广泛,代谢猝灭,均质化,和通过溶剂提取代谢物。由于SPME允许我们在组织样本中的一个动作中执行所有这些步骤,除了其他优点,有必要知道这种方法产生的代谢组和脂质组的覆盖率和性能是否与经典方法相似或相当.
结果:将SPME和固液萃取(Homo-SLE)样品处理方法应用于健康小鼠肾脏组织,其次是全面的代谢组学和脂质组学分析。此外,已经测试了组织的冷冻和储存是否会导致肾脏代谢组和脂质组的改变,因此,对新鲜和冷冻组织样本进行了分析。脂质组学分析显示,在人SLE组中,仅存在不同结构的膜和细胞内脂质。相反,两组均检测到所有带注释的代谢物.值得注意的是,样品的冷冻主要导致大多数脂质种类的水平降低和氨基酸等代谢物的增加,嘌呤,和嘧啶。这些改变主要通过SPME方法以统计学上显著的方式检测。最后,两种方法的样本在所有分析中都显示出正相关。
结论:这些结果表明,在SPME处理中,只要在平衡前动力学状态下进行非详尽提取的基本原理,在组织局部区域提取,考虑到纤维涂层的化学性质和组织的非均匀化,是一种很好的方法,用于肾组织代谢组学;因为这种方法提供了一个易于使用的,高效,和较少侵入性的方法,简化了不同的样品处理步骤。
BACKGROUND: The selection of the sample treatment strategy is a crucial step in the metabolomics workflow. Solid phase microextraction (
SPME) is a sample processing methodology with great potential for use in untargeted metabolomics of tissue samples. However, its utilization is not as widespread as other standard protocols involving steps of tissue collection, metabolism quenching, homogenization, and extraction of metabolites by solvents. Since
SPME allows us to perform all these steps in one action in tissue samples, in addition to other advantages, it is necessary to know whether this methodology produces similar or comparable metabolome and lipidome coverage and performance to classical methods.
RESULTS: SPME and homogenization with solid-liquid extraction (Homo-SLE) sample treatment methods were applied to healthy murine kidney tissue, followed by comprehensive metabolomics and lipidomics analyses. In addition, it has been tested whether freezing and storage of the tissue causes alterations in the renal metabolome and lipidome, so the analyses were performed on fresh and frozen tissue samples Lipidomics analysis revealed the exclusive presence of different structural membrane and intracellular lipids in the Homo-SLE group. Conversely, all annotated metabolites were detected in both groups. Notably, the freezing of the sample mainly causes a decrease in the levels of most lipid species and an increase in metabolites such as amino acids, purines, and pyrimidines. These alterations are principally detected in a statistically significant way by
SPME methodology. Finally, the samples of both methodologies show a positive correlation in all the analyses.
CONCLUSIONS: These results demonstrate that in
SPME processing, as long as the fundamentals of non-exhaustive extraction in a pre-equilibrium kinetic regime, extraction in a tissue localized area, the chemistry of the fiber coating and non-homogenization of the tissue are taken into account, is an excellent method to use in kidney tissue metabolomics; since this methodology presents an easy-to-use, efficient, and less invasive approach that simplifies the different sample processing steps.