Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Advanced Analysis Centre, University of Guelph, Guelph, Ontario, Canada.
Curr Protoc Chem Biol. 2020 Sep;12(3):e83. doi: 10.1002/cpch.83.
Metabolomic studies allow a deeper understanding of the processes of a given ecological community than nucleic acid-based surveys alone. In the case of the gut microbiota, a metabolic profile of, for example, a fecal sample provides details about the function and interactions within the distal region of the gastrointestinal tract, and such a profile can be generated in a number of different ways. This unit elaborates on the use of 1D H NMR spectroscopy as a commonly used method to characterize small-molecule metabolites of the fecal metabonome (meta-metabolome). We describe a set of protocols for the preparation of fecal water extraction, storage, scanning, measurement of pH, and spectral processing and analysis. We also compare the effects of various sample storage conditions for processed and unprocessed samples to provide a framework for comprehensive analysis of small molecules from stool-derived samples. © 2020 Wiley Periodicals LLC Basic Protocol 1: Extracting fecal water from crude fecal samples Alternate Protocol 1: Extracting fecal water from small crude fecal samples Basic Protocol 2: Acquiring NMR spectra of metabolite samples Alternate Protocol 2: Acquiring NMR spectra of metabolite samples using Bruker spectrometer running TopSpin 3.x Alternate Protocol 3: Acquiring NMR spectra of metabolite samples by semiautomated process Basic Protocol 3: Measuring sample pH Support Protocol 1: Cleaning NMR tubes Basic Protocol 4: Processing raw spectra data Basic Protocol 5: Profiling spectra Support Protocol 2: Spectral profiling of sugars and other complex metabolites.
代谢组学研究比仅基于核酸的调查更能深入了解给定生态群落的过程。就肠道微生物组而言，例如粪便样本的代谢特征提供了关于胃肠道远端区域内功能和相互作用的详细信息，并且可以通过多种不同的方式生成这样的图谱。本单元详细介绍了使用 1D H NMR 光谱作为一种常用方法来表征粪便代谢组（元代谢组）中小分子代谢物。我们描述了一组用于准备粪便水提取、储存、扫描、测量 pH 值以及光谱处理和分析的方案。我们还比较了不同处理和未处理样品的各种样品储存条件的影响，为全面分析来自粪便样本的小分子提供了框架。© 2020 威利父子公司 基本方案 1：从粗粪便样品中提取粪便水 替代方案 1：从小的粗粪便样品中提取粪便水 基本方案 2：获取代谢物样品的 NMR 图谱 替代方案 2：使用 Bruker 光谱仪运行 TopSpin 3.x 获取代谢物样品的 NMR 图谱 替代方案 3：通过半自动过程获取代谢物样品的 NMR 图谱 基本方案 3：测量样品 pH 值 支持方案 1：清洁 NMR 管 基本方案 4：处理原始光谱数据 基本方案 5：分析光谱 支持方案 2：糖和其他复杂代谢物的光谱分析。
