Absolute metabolite concentrations are essential information for quantitative metabolomics studies, as concentrations are closely related to metabolic reactions, enzyme kinetics and other important biological activities. A well-performed metabolites extraction procedure, a reliable detection method, and a robust quantitative approach are all critical factors for obtaining absolute metabolite concentrations. Here, we used a HPLC-MS/MS based platform to successfully develop a 13C-labeled quantitative metabolomics approach, and applied this novel method to quantify bacterial metabolite concentrations in three different domains (i.e., intracellularly, extracellularly and to the whole culture), with high accuracy for a model Escherichia coli bacteria. The bacterial culture was grown in universal 13C-labeled medium and the metabolites were extracted by 40/40/20/methanol/ACN/H2O with 0.1% formic acid. One hundred and twenty-five metabolites were initially screened and one hundred and six 13C metabolites were confidently detected from the model bacteria grown in 13C-labeled medium. A subset of twenty-one metabolites was subsequently quantified using 12C-metabolite chemical standards to assist the calculation of 13C metabolite concentration. This rigorous 13C-labled quantitative method was then applied to characterize the metabolic profile changes in three domains of E. coli going through antibiotic treatment. Our results demonstrated that metabolites from all three domains can be used to significantly differentiate the ampicillin treatment group and control group (without ampicillin). In conclusion, our work demonstrated that the quantitative metabolomics approach can be used as a valuable tool to study bacterial metabolism in different domains and to understand their response to environmental perturbations.

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不同结构域细菌代谢物的定量代谢组学研究

绝对代谢物浓度是定量代谢组学研究的重要信息，因为浓度与代谢反应、酶动力学和其他重要的生物活性密切相关。执行良好的代谢物提取程序、可靠的检测方法和可靠的定量方法都是获得绝对代谢物浓度的关键因素。在这里，我们使用基于 HPLC-MS/MS 的平台成功开发了 13C 标记的定量代谢组学方法，并应用这种新方法来量化三个不同域（即细胞内、细胞外和整个培养物）中的细菌代谢物浓度，对模型大肠杆菌具有高精度。细菌培养物在通用 13C 标记培养基中生长，代谢物用 40/40/20/甲醇/乙腈/水和 0.1% 甲酸提取。最初筛选了 125 种代谢物，并从 13C 标记培养基中生长的模型细菌中可靠地检测到了 106 种 13C 代谢物。随后使用 12C 代谢物化学标准对 21 种代谢物的子集进行量化，以帮助计算 13C 代谢物浓度。然后应用这种严格的 13C 标记的定量方法来表征经过抗生素治疗的大肠杆菌三个域的代谢谱变化。我们的结果表明，来自所有三个域的代谢物可用于显着区分氨苄青霉素治疗组和对照组（不含氨苄青霉素）。