Xu Wei, Chen Deying, Wang Nan, Zhang Ting, Zhou Ruokun, Huan Tao, Lu Yingfeng, Su Xiaoling, Xie Qing, Li Liang, Li Lanjuan
State Key Laboratory and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University , Hangzhou 310003, China.
Anal Chem. 2015 Jan 20;87(2):829-36. doi: 10.1021/ac503619q. Epub 2014 Dec 25.
Human fecal samples contain endogenous human metabolites, gut microbiota metabolites, and other compounds. Profiling the fecal metabolome can produce metabolic information that may be used not only for disease biomarker discovery, but also for providing an insight about the relationship of the gut microbiome and human health. In this work, we report a chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method for comprehensive and quantitative analysis of the amine- and phenol-containing metabolites in fecal samples. Differential (13)C2/(12)C2-dansyl labeling of the amines and phenols was used to improve LC separation efficiency and MS detection sensitivity. Water, methanol, and acetonitrile were examined as an extraction solvent, and a sequential water-acetonitrile extraction method was found to be optimal. A step-gradient LC-UV setup and a fast LC-MS method were evaluated for measuring the total concentration of dansyl labeled metabolites that could be used for normalizing the sample amounts of individual samples for quantitative metabolomics. Knowing the total concentration was also useful for optimizing the sample injection amount into LC-MS to maximize the number of metabolites detectable while avoiding sample overloading. For the first time, dansylation isotope labeling LC-MS was performed in a simple time-of-flight mass spectrometer, instead of high-end equipment, demonstrating the feasibility of using a low-cost instrument for chemical isotope labeling metabolomics. The developed method was applied for profiling the amine/phenol submetabolome of fecal samples collected from three families. An average of 1785 peak pairs or putative metabolites were found from a 30 min LC-MS run. From 243 LC-MS runs of all the fecal samples, a total of 6200 peak pairs were detected. Among them, 67 could be positively identified based on the mass and retention time match to a dansyl standard library, while 581 and 3197 peak pairs could be putatively identified based on mass match using MyCompoundID against a Human Metabolome Database and an Evidence-based Metabolome Library, respectively. This represents the most comprehensive profile of the amine/phenol submetabolome ever detected in human fecal samples. The quantitative metabolome profiles of individual samples were shown to be useful to separate different groups of samples, illustrating the possibility of using this method for fecal metabolomics studies.
人类粪便样本包含内源性人类代谢物、肠道微生物群代谢物及其他化合物。分析粪便代谢组可产生代谢信息,这些信息不仅可用于发现疾病生物标志物,还能深入了解肠道微生物群与人类健康的关系。在本研究中,我们报告了一种化学同位素标记液相色谱 - 质谱联用(LC - MS)方法,用于全面定量分析粪便样本中含胺和含酚的代谢物。采用胺和酚的差分(13)C2/(12)C2 - 丹磺酰基标记来提高LC分离效率和MS检测灵敏度。考察了水、甲醇和乙腈作为萃取溶剂,发现顺序水 - 乙腈萃取法最为理想。评估了一种梯度LC - UV设置和一种快速LC - MS方法,用于测量丹磺酰基标记代谢物的总浓度,该浓度可用于定量代谢组学中对各个样本的样本量进行归一化。了解总浓度对于优化进样到LC - MS中的样本量也很有用,既能最大限度地检测到可检测代谢物的数量,又能避免样本过载。首次在简单的飞行时间质谱仪而非高端设备上进行丹磺酰化同位素标记LC - MS,证明了使用低成本仪器进行化学同位素标记代谢组学的可行性。所开发的方法应用于分析从三个家庭收集的粪便样本的胺/酚亚代谢组。在30分钟的LC - MS运行中平均发现1785个峰对或推定代谢物。在所有粪便样本的243次LC - MS运行中,总共检测到6200个峰对。其中,67个峰对可根据与丹磺酰基标准库的质量和保留时间匹配得到明确鉴定,而分别使用MyCompoundID与人类代谢组数据库和基于证据的代谢组库进行质量匹配时,可推定鉴定出581个和3197个峰对。这代表了在人类粪便样本中检测到的最全面的胺/酚亚代谢组图谱。各个样本的定量代谢组图谱显示有助于区分不同组别的样本,说明了使用该方法进行粪便代谢组学研究的可能性。