Pseudotargeted metabolomics: A method for semi-quantitatively monitoring thousand of metabolites

We developed a pseudotargeted metabolomics method, ion-pairs used for MRM are extracted from untargeted mass spectrometry data by using mixtures of samples to be analyzed, semi-quantitative information of each sample is obtained by the MRM without identification of the metabolites.

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Metabolomics is the science of studying endogenous metabolites. The concentrations and variation of metabolites can offer fresh insight into biological processes. The dream of scientists who focus on metabolomics analysis is to comprehensively characterize all metabolites in biological samples both qualitatively and quantitatively. But it’s very difficult to achieve this aim because of metabolome complexity1.

Untargeted method based on high-resolution mass spectrometry (HRMS) is the most typical method in metabolomics research. It is non-biased with rich information, but suffers from complex data handling, poor repeatability, and narrow linear range. Interestingly, the targeted method based on triple quadrupole mass spectrometry (TQMS) does not have these shortcomings, unfortunately, usually only for known metabolites. So targeted detection on TQMS with a similar metabolite coverage to the untargeted method is a wish for the scientists in this field. To realize this aim, we have developed a novel metabolomics method named as “pseudotargeted method”2, it merges the advantages of untargeted and targeted metabolomics methods (Figure 1).

Figure 1. Pseudotargeted metabolomics method is a new method integrating the advantages of untargeted and targeted metabolomics methods.

A key step for establishing a pseudotargeted method is to obtain the ion-pairs of metabolites in samples to be analyzed for multi-reaction monitoring (MRM) detection of TQMS, it is time-costing and not easy. This limits the promotion and application of the pseudotargeted method in metabolomics. So we have embarked on creating a standard protocol for the pseudotargeted metabolomics3 (Figure 2), and hope that the technique becomes more accessible to other researchers, and let more people use the pseudotargeted method to study the metabolism and metabolites. 


Figure 2. The workflow of pseudotargeted method

To define MRM ion-pairs automatically and systematically, the in-house software“Multiple Reaction Monitoring-Ion Pair Finder (MRM-Ion Pair Finder)”based on the Matlab was developed4, which made defining of the MRM ion-pairs for untargeted metabolic profiling easier and less time consuming. However, the Matlab is not free, and some researchers may not use it. In contrast, R provides a free software environment for statistical computing and graphics and is very popular among researchers. So, we have reprogrammed our MRM-Ion Pair Finder in R to provide more choices for researchers3.

Another question often asked is about the universality of the pseudotargeted method in different mass spectrometry instruments. To address this issue, we have used the same sample and implemented pseudotargeted methods on three different TQMS instruments. Excitingly, the pseudotargeted methods implemented on different instruments are highly consistent. No matter what kind of TQMS is, similar pseudotargeted method can be achieved3.

In summary, pseudotargeted method is a stable, high coverage semi-quantitative method, and could be an alternative of untargeted method. We believe that pseudotargeted method can be a powerful tool for the metabolomics researches. We welcome more scientists to try and to use the pseudotargeted method to solve their actual problems. If you have any technical problems, please feel free to contact us.

Written by Fujian Zheng, Xinjie Zhao & Guowang Xu


  1. N Blow (2008) Metabolomics: Biochemistry's new look, Nature, 455:697-698
  2. Shili Chen, Hongwei Kong, Xin Lu, Yong Li, Peiyuan Yin, Zhongda Zeng, Guowang Xu*(2013) Pseudotargeted Metabolomics Method and Its Application in Serum Biomarker Discovery for Hepatocellular Carcinoma Based on Ultra High-Performance Liquid Chromatography/Triple Quadrupole Mass Spectrometry, Anal. Chem. 85(17):8326-33
  3. Fujian Zheng, Xinjie Zhao, Zhongda Zeng, Lichao Wang, Wangjie Lv, Qingqing Wang, Guowang Xu* (2020) Development of plasma pseudotargeted metabolomics method based on ultra-high performance liquid chromatography-mass spectrometry, Nature Protocols, accepted
  4. Ping Luo,Weidong Dai, Peiyuan Yin, Zhongda Zeng, Lina Zhou, Xiaolin Wang, Shili Chen, Xin Lu, Guowang Xu* (2015) Multiple Reaction Monitoring-Ion Pair Finder: A Systematic Approach To Transform Nontargeted Mode to Pseudotargeted Mode for Metabolomics Study Based on Liquid Chromatography–Mass Spectrometry, Anal. Chem., 87 (10): 5050–5055
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Fujian Zheng

PHD student, Dalian Institute of Chemical Physics

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