Isotopic Ratio Outlier Analysis Global Metabolomics of Caenorhabditis elegans

We demonstrate the global metabolic analysis of Caenorhabditis elegans stress responses using a mass-spectrometry-based technique called isotopic ratio outlier analysis (IROA). In an IROA protocol, control and experimental samples are isotopically labeled with 95 and 5% 13C, and the two sample populations are mixed together for uniform extraction, sample preparation, and LC-MS analysis. This labeling strategy provides several advantages over conventional approaches: (1) compounds arising from biosynthesis are easily distinguished from artifacts, (2) errors from sample extraction and preparation are minimized because the control and experiment are combined into a single sample, (3) measurement of both the molecular weight and the exact number of carbon atoms in each molecule provides extremely accurate molecular formulas, and (4) relative concentrations of all metabolites are easily determined. A heat-shock perturbation was conducted on C. elegans to demonstrate this approach. We identified many compounds that significantly changed upon heat shock, including several from the purine metabolism pathway. The metabolomic response information by IROA may be interpreted in the context of a wealth of genetic and proteomic information available for C. elegans. Furthermore, the IROA protocol can be applied to any organism that can be isotopically labeled, making it a powerful new tool in a global metabolomics pipeline.本研究采用基于质谱的同位素比值异常分析（isotopic ratio outlier analysis，IROA）技术，开展秀丽隐杆线虫（Caenorhabditis elegans）应激反应的全局代谢组分析。在IROA实验流程中，对照组与实验组样本分别以95%和5%的¹³C进行同位素标记，随后将两类样本混合，以实现统一的样品提取、前处理及液相色谱-质谱（LC-MS）分析。相较于传统方法，该标记策略具备多重优势：（1）生物合成产生的化合物可与实验伪迹轻松区分；（2）由于对照组与实验组合并为单一样本，样品提取与前处理环节引入的误差可被最小化；（3）同时测定每个分子的分子量与精确碳原子数目，可得到精度极高的分子式；（4）所有代谢物的相对浓度均可便捷定量。本研究通过对秀丽隐杆线虫实施热激扰动实验，验证了该方法的有效性。研究鉴定出热激后发生显著变化的多种化合物，其中包括嘌呤代谢通路中的多个组分。依托秀丽隐杆线虫已有的海量遗传与蛋白质组学数据，可对IROA技术获取的代谢组响应信息进行系统性解读。此外，IROA实验流程可推广至任何可进行同位素标记的生物，使其成为全局代谢组学研究流程中的一款高效新型工具。