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Liquid chromatography quadrupole time-of-flight mass spectrometry characterization of metabolites guided by the METLIN database

Nature Protocols volume 8, pages 451–460 (2013)Cite this article

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Abstract

Untargeted metabolomics provides a comprehensive platform for identifying metabolites whose levels are altered between two or more populations. By using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS), hundreds to thousands of peaks with a unique m/z ratio and retention time are routinely detected from most biological samples in an untargeted profiling experiment. Each peak, termed a metabolomic feature, can be characterized on the basis of its accurate mass, retention time and tandem mass spectral fragmentation pattern. Here a seven-step protocol is suggested for such a characterization by using the METLIN metabolite database. The protocol starts from untargeted metabolomic LC-Q-TOF-MS data that have been analyzed with the bioinformatics program XCMS, and it describes a strategy for selecting interesting features as well as performing subsequent targeted tandem MS. The seven steps described will require 2–4 h to complete per feature, depending on the compound.

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Figure 1: Determination of monoisotopic peak, charge state and adduct of the precursor ion.
Figure 2: Insufficient chromatographic resolution of a species can lead to overlapping peaks that produce convoluted MS/MS spectra.
Figure 3: Screenshot of metabolite search in METLIN.
Figure 4
Figure 5: Screenshot of the spectrum of hypoxanthine.
Figure 6: A comparison of experimental (black) and METLIN standard (red) spectra for three metabolites.
Figure 7: The importance of retention time, accurate mass and fragmentation for identification.

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Acknowledgements

This work was supported by the California Institute of Regenerative Medicine (no. TR1-01219) (G.S.), the US National Institutes of Health (nos. R01 CA170737 (G.S.), R24 EY017540 (G.S.), P30 MH062261 (G.S.), RC1 HL101034(G.S.), P01 DA026146 (G.S.), and 1R01 ES022181-01) (G.J.P.) and the US National Institutes of Health-National Institute on Aging (no. L30 AG0 038036) (G.J.P.). Financial support was also received from the US Department of Energy (grant nos. FG02-07ER64325 and DE-AC0205CH11231) (G.S.).

Author information

Authors and Affiliations

  1. Scripps Center for Metabolomics and Mass Spectrometry, The Scripps Research Institute, La Jolla, California, USA

    Zheng-Jiang Zhu, Andrew W Schultz, Junhua Wang, Caroline H Johnson & Gary Siuzdak

  2. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Steven M Yannone

  3. Department of Chemistry, Washington University, St. Louis, Missouri, USA

    Gary J Patti

  4. Department of Genetics, Washington University, St. Louis, Missouri, USA

    Gary J Patti

  5. Department of Medicine, Washington University, St. Louis, Missouri, USA

    Gary J Patti

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  1. Zheng-Jiang Zhu
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Contributions

Z.-J.Z., A.W.S. and J.W. and contributed equally to the work described. G.J.P. and G.S. supervised the work. A.W.S., J.W. and G.J.P. performed the experiments. Z.-J.Z., A.W.S., J.W. and C.H.J. wrote the manuscript. Z.-J.Z., S.M.Y., G.J.P. and G.S. read and revised the manuscript.

Corresponding authors

Correspondence to Gary J Patti or Gary Siuzdak.

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Supplementary information

Supplementary Figure 1

Example of in-source fragmentation. Two species, m/z 480.3084 and m/z 339.2892 are observed to coelute (A), with both species observed in MS (B). Comparison of the high resolution parent ion and the MS/MS fragment (D) supports the characterization of m/z 480.3084 as a lysoPE(18:1). Note the prominent fragment at m/z 339.2892. m/z 339.2892 is observed in the MS scan (D). Fragmentation of this species provides the MS/MS spectrum (C) which is characteristic of a dehydrated oleoyl (18:1) glycerol, a major fragment of lysoPE(18:1). (PDF 411 kb)

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Zhu, ZJ., Schultz, A., Wang, J. et al. Liquid chromatography quadrupole time-of-flight mass spectrometry characterization of metabolites guided by the METLIN database. Nat Protoc 8, 451–460 (2013). https://doi.org/10.1038/nprot.2013.004

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