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Development of a plasma pseudotargeted metabolomics method based on ultra-high-performance liquid chromatography–mass spectrometry

Abstract

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.

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Fig. 1: Schematic of the retention-time calibration method.
Fig. 2
Fig. 3: MRM transitions verified by different TQMS.
Fig. 4: Quantitative performance of the pseudotargeted metabolomics method.

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Acknowledgements

The study is supported by the foundations (21435006 and 21974139) from the National Natural Science Foundation of China, the National Key Research and Development Program of China (2017YFC0906900) and the innovation program (DICP TMSR201601) of science and research from the DICP, CAS.

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Contributions

G.X. and X.Z. developed the concept, designed experiments and optimized the manuscript. F.Z. performed the experiments, analyzed the data and wrote the manuscript. Z.Z. provided the software named ‘MRM-Ion Pair Finder’, and F.Z. modified this software. L.W., W.L. and Q.W. gave technical support and conceptual advice.

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Correspondence to Guowang Xu.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Thomas Wilson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Li, Y., et al. J. Chromatogr. A 1255, 228–236 (2012): https://www.sciencedirect.com/science/article/abs/pii/S0021967312002026

Chen, S., et al. Anal. Chem. 85, 8326–8333 (2013): https://pubs.acs.org/doi/10.1021/ac4016787

Luo, P., et al. Anal. Chem. 87, 5050–5055 (2015): https://pubs.acs.org/doi/10.1021/acs.analchem.5b00615

Shao, Y., et al. J. Proteome Res. 14, 906–916 (2015): https://pubs.acs.org/doi/10.1021/pr500973d

Luo, P., et al. Hepatology 67, 662–675 (2018): https://aasldpubs.onlinelibrary.wiley.com/doi/abs/10.1002/hep.29561

Key data used in this protocol

Luo, P., et al. Anal. Chem. 87, 5050–5055 (2015): https://pubs.acs.org/doi/10.1021/acs.analchem.5b00615

Supplementary information

Supplementary Information

Supplementary Figures 1–3 and Supplementary Tables 1, 2 and 5.

Reporting Summary

Supplementary Table 3

MRM transitions from NIST SRM 1950 in positive ion mode.

Supplementary Table 4

MRM transitions from NIST SRM 1950 in negative ion mode.

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Zheng, F., Zhao, X., Zeng, Z. et al. Development of a plasma pseudotargeted metabolomics method based on ultra-high-performance liquid chromatography–mass spectrometry. Nat Protoc 15, 2519–2537 (2020). https://doi.org/10.1038/s41596-020-0341-5

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