Zheng Fujian, Zhao Xinjie, Zeng Zhongda, Wang Lichao, Lv Wangjie, Wang Qingqing, Xu Guowang
CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
University of Chinese Academy of Sciences, Beijing, China.
Nat Protoc. 2020 Aug;15(8):2519-2537. doi: 10.1038/s41596-020-0341-5. Epub 2020 Jun 24.
Untargeted methods are typically used in the detection and discovery of small organic compounds in metabolomics research, and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) is one of the most commonly used platforms for untargeted metabolomics. Although they are non-biased and have high coverage, untargeted approaches suffer from unsatisfying repeatability and a requirement for complex data processing. Targeted metabolomics based on triple-quadrupole mass spectrometry (TQMS) could be a complementary tool because of its high sensitivity, high specificity and excellent quantification ability. However, it is usually applicable to known compounds: compounds whose identities are known and/or are expected to be present in the analyzed samples. Pseudotargeted metabolomics merges the advantages of untargeted and targeted metabolomics and can act as an alternative to the untargeted method. Here, we describe a detailed protocol of pseudotargeted metabolomics using UHPLC-TQMS. An in-depth, untargeted metabolomics experiment involving multiple UHPLC-HRMS runs with MS at different collision energies (both positive and negative) is performed using a mixture obtained using small amounts of the analyzed samples. XCMS, CAMERA and Multiple Reaction Monitoring (MRM)-Ion Pair Finder are used to find and annotate peaks and choose transitions that will be used to analyze the real samples. A set of internal standards is used to correct for variations in retention time. High coverage and high-performance quantitative analysis can be realized. The entire protocol takes ~5 d to complete and enables the simultaneously semiquantitative analysis of 800-1,300 metabolites.
非靶向方法通常用于代谢组学研究中有机小分子化合物的检测与发现，超高效液相色谱-高分辨率质谱联用仪（UHPLC-HRMS）是最常用的非靶向代谢组学平台之一。尽管非靶向方法无偏向性且覆盖范围广，但存在重复性不尽人意以及需要复杂数据处理的问题。基于三重四极杆质谱（TQMS）的靶向代谢组学因其高灵敏度、高特异性和出色的定量能力，可作为一种补充工具。然而，它通常适用于已知化合物：即在分析样品中其身份已知和/或预期存在的化合物。伪靶向代谢组学融合了非靶向和靶向代谢组学的优点，可作为非靶向方法的替代方案。在此，我们描述了一种使用UHPLC-TQMS进行伪靶向代谢组学的详细方案。使用由少量分析样品获得的混合物，进行深入的非靶向代谢组学实验，该实验涉及多次不同碰撞能量（正离子和负离子模式）下的UHPLC-HRMS运行。利用XCMS、CAMERA和多反应监测（MRM）-离子对查找器来查找和注释峰，并选择用于分析实际样品的跃迁。使用一组内标来校正保留时间的变化。可实现高覆盖度和高性能的定量分析。整个方案大约需要5天完成，能够同时对800 - 1300种代谢物进行半定量分析。
