定量代谢组学的方法构建及其在肠道微生态研究中的应用
发布时间：2018-10-15 10:10
【摘要】：人类粪便样本中主要包括人体内源性代谢物,肠道菌群代谢物及其他化合物。对于粪便代谢组的分析不仅可以为发现疾病生物标志提供代谢信息,同时也可以为分析肠道菌群及人类健康间的关系提供新的切入点。在本研究中,我们使用化学同位素标记高效液相色谱-质谱(HPLC-MS)的方法完整并定量地分析了人类粪便样本中含胺及酚的代谢物,并利用13C2/12C2-丹璜酰区分标记胺和酚以提高色谱分析效率及质谱检测灵敏度。在本研究中,水,甲醇和乙腈被用作提取溶剂,而水→乙腈梯度提取表现出的最佳的提取效率。在定量研究中,我们使用梯度液相色谱-紫外及快速液相色谱-质谱检测对单个样本代谢组进行标准化。同时,明确单个样本总浓度有助于优化样本的最佳进样量,这样可以最大化液相色谱-质谱检测中可检测代谢物的数量,并避免分析柱过载。本研究首次利用简单的飞行时间质谱仪进行丹磺酰标记的液相色谱-质谱分析,说明利用低成本仪器进行化学同位素标记代谢组学分析是可行的。样本是从三个家庭中采集的粪便,并利用上述方法进行胺/酚亚代谢组成分分析。结果显示,30分钟的色谱-质谱分析平均能检测出1785个峰或推定代谢物,而243次色谱-质谱分析共检测出6200个峰。其中,67个峰通过将其分子质量及保留时间与丹磺酰标准库进行匹配而成功鉴定。此外,利用MyCompoundID所收录的数据,581个峰通过将其分子量与人类代谢组数据库进行匹配而推定,3197个峰通过将其分子量与循证代谢组数据库进行匹配而推定。对单个样本的定量代谢组分析显示其能够有效区分不同组别的样本,提示该方法可能用于之后的粪便代谢组研究中。
[Abstract]:Human fecal samples mainly include endogenous metabolites, intestinal flora metabolites and other compounds. The analysis of fecal metabolites can not only provide metabolic information for the discovery of disease biomarkers, but also provide a new entry point for analyzing the relationship between intestinal flora and human health. In this study, we used chemical isotope labeled high performance liquid chromatography-mass spectrometry (HPLC-MS) to analyze the metabolites of amines and phenols in human fecal samples. 13C _ 2 / 12C _ 2-Danhuanyl was used to distinguish labeled amines and phenols to improve the efficiency of chromatographic analysis and the sensitivity of mass spectrometry. In this study, water, methanol and acetonitrile were used as solvent, and the gradient extraction of water acetonitrile showed the best extraction efficiency. In quantitative analysis, gradient liquid chromatography-ultraviolet (GLC) and rapid liquid chromatography-mass spectrometry (RLC-MS) were used to standardize the metabolism of a single sample. At the same time, the determination of the total concentration of a single sample is helpful to optimize the optimal sample size, which can maximize the quantity of metabolites detected in liquid chromatography-mass spectrometry (LC-MS) and avoid the analysis column overload. In this study, a simple time-of-flight mass spectrometer (TTOMS) was used for the analysis of Dansulfonyl labeled by liquid chromatography-mass spectrometry (LC-MS), which indicated that it is feasible to use a low-cost instrument to analyze the metabolomics of chemical isotope labeling. The samples were collected from three families and the amines / phenols submetabolites were analyzed by the above method. The results showed that an average of 1785 peaks or presumed metabolites could be detected by 30-minute chromatography-mass spectrometry analysis, while 6200 peaks were detected by 243-time chromatography-mass spectrometry analysis. Among them, 67 peaks were successfully identified by matching the molecular weight and retention time with the Dansulfonyl standard library. In addition, using the data collected by MyCompoundID, 581 peaks were inferred by matching their molecular weight with the human metabolic group database, and 3197 peaks by matching their molecular weight with the evidence-based metabolic group database. Quantitative metabonomics analysis of a single sample shows that it can effectively distinguish samples from different groups, suggesting that this method may be used in later studies of fecal metabolites.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.13
[Abstract]:Human fecal samples mainly include endogenous metabolites, intestinal flora metabolites and other compounds. The analysis of fecal metabolites can not only provide metabolic information for the discovery of disease biomarkers, but also provide a new entry point for analyzing the relationship between intestinal flora and human health. In this study, we used chemical isotope labeled high performance liquid chromatography-mass spectrometry (HPLC-MS) to analyze the metabolites of amines and phenols in human fecal samples. 13C _ 2 / 12C _ 2-Danhuanyl was used to distinguish labeled amines and phenols to improve the efficiency of chromatographic analysis and the sensitivity of mass spectrometry. In this study, water, methanol and acetonitrile were used as solvent, and the gradient extraction of water acetonitrile showed the best extraction efficiency. In quantitative analysis, gradient liquid chromatography-ultraviolet (GLC) and rapid liquid chromatography-mass spectrometry (RLC-MS) were used to standardize the metabolism of a single sample. At the same time, the determination of the total concentration of a single sample is helpful to optimize the optimal sample size, which can maximize the quantity of metabolites detected in liquid chromatography-mass spectrometry (LC-MS) and avoid the analysis column overload. In this study, a simple time-of-flight mass spectrometer (TTOMS) was used for the analysis of Dansulfonyl labeled by liquid chromatography-mass spectrometry (LC-MS), which indicated that it is feasible to use a low-cost instrument to analyze the metabolomics of chemical isotope labeling. The samples were collected from three families and the amines / phenols submetabolites were analyzed by the above method. The results showed that an average of 1785 peaks or presumed metabolites could be detected by 30-minute chromatography-mass spectrometry analysis, while 6200 peaks were detected by 243-time chromatography-mass spectrometry analysis. Among them, 67 peaks were successfully identified by matching the molecular weight and retention time with the Dansulfonyl standard library. In addition, using the data collected by MyCompoundID, 581 peaks were inferred by matching their molecular weight with the human metabolic group database, and 3197 peaks by matching their molecular weight with the evidence-based metabolic group database. Quantitative metabonomics analysis of a single sample shows that it can effectively distinguish samples from different groups, suggesting that this method may be used in later studies of fecal metabolites.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.13
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