【摘要】：人類糞便樣本中主要包括人體內(nèi)源性代謝物,腸道菌群代謝物及其他化合物。對于糞便代謝組的分析不僅可以為發(fā)現(xiàn)疾病生物標(biāo)志提供代謝信息,同時也可以為分析腸道菌群及人類健康間的關(guān)系提供新的切入點(diǎn)。在本研究中,我們使用化學(xué)同位素標(biāo)記高效液相色譜-質(zhì)譜(HPLC-MS)的方法完整并定量地分析了人類糞便樣本中含胺及酚的代謝物,并利用13C2/12C2-丹璜酰區(qū)分標(biāo)記胺和酚以提高色譜分析效率及質(zhì)譜檢測靈敏度。在本研究中,水,甲醇和乙腈被用作提取溶劑,而水→乙腈梯度提取表現(xiàn)出的最佳的提取效率。在定量研究中,我們使用梯度液相色譜-紫外及快速液相色譜-質(zhì)譜檢測對單個樣本代謝組進(jìn)行標(biāo)準(zhǔn)化。同時,明確單個樣本總濃度有助于優(yōu)化樣本的最佳進(jìn)樣量,這樣可以最大化液相色譜-質(zhì)譜檢測中可檢測代謝物的數(shù)量,并避免分析柱過載。本研究首次利用簡單的飛行時間質(zhì)譜儀進(jìn)行丹磺酰標(biāo)記的液相色譜-質(zhì)譜分析,說明利用低成本儀器進(jìn)行化學(xué)同位素標(biāo)記代謝組學(xué)分析是可行的。樣本是從三個家庭中采集的糞便,并利用上述方法進(jìn)行胺/酚亞代謝組成分分析。結(jié)果顯示,30分鐘的色譜-質(zhì)譜分析平均能檢測出1785個峰或推定代謝物,而243次色譜-質(zhì)譜分析共檢測出6200個峰。其中,67個峰通過將其分子質(zhì)量及保留時間與丹磺酰標(biāo)準(zhǔn)庫進(jìn)行匹配而成功鑒定。此外,利用MyCompoundID所收錄的數(shù)據(jù),581個峰通過將其分子量與人類代謝組數(shù)據(jù)庫進(jìn)行匹配而推定,3197個峰通過將其分子量與循證代謝組數(shù)據(jù)庫進(jìn)行匹配而推定。對單個樣本的定量代謝組分析顯示其能夠有效區(qū)分不同組別的樣本,提示該方法可能用于之后的糞便代謝組研究中。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.13

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