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Using MetaboAnalyst 5.0 for LC–HRMS spectra processing, multi-omics integration and covariate adjustment of global metabolomics data

Nature Protocols volume 17, pages 1735–1761 (2022)Cite this article

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Abstract

Liquid chromatography coupled with high-resolution mass spectrometry (LC–HRMS) has become a workhorse in global metabolomics studies with growing applications across biomedical and environmental sciences. However, outstanding bioinformatics challenges in terms of data processing, statistical analysis and functional interpretation remain critical barriers to the wider adoption of this technology. To help the user community overcome these barriers, we have made major updates to the well-established MetaboAnalyst platform (www.metaboanalyst.ca). This protocol extends the previous 2011 Nature Protocol by providing stepwise instructions on how to use MetaboAnalyst 5.0 to: optimize parameters for LC–HRMS spectra processing; obtain functional insights from peak list data; integrate metabolomics data with transcriptomics data or combine multiple metabolomics datasets; conduct exploratory statistical analysis with complex metadata. Parameter optimization may take ~2 h to complete depending on the server load, and the remaining three stages may be executed in ~60 min.

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Fig. 1: MetaboAnalyst 5.0 overview.
Fig. 2: A screenshot of the result download page.
Fig. 3: Results from raw spectra processing.
Fig. 4: Functional analysis of ranked peak lists.
Fig. 5: Functional analysis of manually selected metabolic patterns.
Fig. 6: Results of joint-pathway analysis.
Fig. 7: Graphical results of functional meta-analysis.
Fig. 8: Metadata correlations and associations with metabolomics data.
Fig. 9: Results of covariate adjustment.

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Data availability

All example datasets used in the protocol are integrated as example datasets in their respective modules and are also available for download from the ‘Format’ page of MetaboAnalyst (https://www.metaboanalyst.ca/MetaboAnalyst/docs/Format.xhtml). There are no restrictions on their use.

Code availability

MetaboAnalyst is freely accessible as a web-based application. The underlying R code is freely available at GitHub as the MetaboAnalystR (https://github.com/xia-lab/MetaboAnalystR) and OptiLCMS (https://github.com/xia-lab/OptiLCMS) packages under the GNU General Public License version 2 or later.

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Acknowledgements

We thank Genome Canada, Génome Québec, US National Institutes of Health (U01 CA235493), Natural Sciences and Engineering Research Council of Canada (NSERC) and Canada Research Chairs (CRC) Program for funding support.

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Authors and Affiliations

  1. Institute of Parasitology, McGill University, Montreal, Quebec, Canada

    Zhiqiang Pang, Guangyan Zhou, Orcun Hacariz & Jianguo Xia

  2. Department of Natural Resources Sciences, McGill University, Montreal, Quebec, Canada

    Jessica Ewald & Niladri Basu

  3. Department of Human Genetics, McGill University, Montreal, Quebec, Canada

    Le Chang & Jianguo Xia

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  1. Zhiqiang Pang
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  4. Le Chang
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Contributions

Z.P., J.E., N.B. and J.X. prepared the manuscript. Z.P., G.Z., J.E., L.C., O.H. and J.X. contributed to the development and testing of the MetaboAnalyst. All authors read and approved the final manuscript.

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Correspondence to Jianguo Xia.

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Nature Protocols thanks Julia Kuligowski, Zhenzhen Xu 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

Pang, Z. et al. Metabolites 10, 186 (2020): https://doi.org/10.3390/metabo10050186

Pang, Z. et al. Metabolites 11, 44 (2021): https://doi.org/10.3390/metabo11010044

Pang, Z. et al. Nucleic Acids Res. 49, W388–W396 (2021): https://doi.org/10.1093/nar/gkab382

Key data used in this protocol

Gardinassi, L. G. et al. Redox Biol. 17, 158–170 (2018): https://doi.org/10.1016/j.redox.2018.04.011

Walker, D. I. et al. Int. J. Epidemiol. 45, 1517–1527 (2016): https://doi.org/10.1093/ije/dyw218

This protocol is an extension to: Nat. Protoc. 6, 743–760 (2011): https://doi.org/10.1038/nprot.2011.319.

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Supplementary Figs. 1 and 2 and Supplementary Table 1.

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Pang, Z., Zhou, G., Ewald, J. et al. Using MetaboAnalyst 5.0 for LC–HRMS spectra processing, multi-omics integration and covariate adjustment of global metabolomics data. Nat Protoc 17, 1735–1761 (2022). https://doi.org/10.1038/s41596-022-00710-w

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