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Global metabolic profiling of animal and human tissues via UPLC-MS

Nature Protocols volume 8, pages 17–32 (2013)Cite this article

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Abstract

Obtaining comprehensive, untargeted metabolic profiles for complex solid samples, e.g., animal tissues, requires sample preparation and access to information-rich analytical methodologies such as mass spectrometry (MS). Here we describe a practical two-step process for tissue samples that is based on extraction into 'aqueous' and 'organic' phases for polar and nonpolar metabolites. Separation methods such as ultraperformance liquid chromatography (UPLC) in combination with MS are needed to obtain sufficient resolution to create diagnostic metabolic profiles and identify candidate biomarkers. We provide detailed protocols for sample preparation, chromatographic procedures, multivariate analysis and metabolite identification via tandem MS (MS/MS) techniques and high-resolution MS. By using these optimized approaches, analysis of a set of samples using a 96-well plate format would take ∼48 h: 1 h for system setup, 8–10 h for sample preparation, 34 h for UPLC-MS analysis and 2–3 h for preliminary/exploratory data processing, representing a robust method for untargeted metabolic screening of tissue samples.

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Figure 1
Figure 2
Figure 3
Figure 4: Typical UPLC-MS base peak ion (BPI) chromatograms of extracted liver samples from rat.
Figure 5: PC1 versus PC2 scores plot for aqueous rat liver extracts analyzed by UPLC-MS.

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Acknowledgements

E.J.W. acknowledges funding support from Waters Corporation.

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Authors and Affiliations

  1. Department of Surgery and Cancer, Biomolecular Medicine, Faculty of Medicine, Imperial College, London, UK

    Elizabeth J Want, Perrine Masson, Ian D Wilson, Elaine Holmes & Jeremy K Nicholson

  2. Department of Chemistry, Laboratory of Analytical Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece

    Filippos Michopoulos & Georgios Theodoridis

  3. Pharmaceutical Business Operations, Waters Corporation, Milford, Massachusetts, USA

    Robert S Plumb & John Shockcor

  4. Mass Spectrometry Business Unit, Shimadzu Corporation, Manchester, UK

    Neil Loftus

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  1. Elizabeth J Want
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Contributions

All authors discussed the design of the manuscript, including the protocol steps and anticipated results. E.J.W. and I.D.W. wrote the manuscript, P.M. designed much of the protocol with E.J.W. G.T., R.S.P., J.S., F.M., N.L. and E.H. made valuable contributions and J.K.N. revised the manuscript.

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Correspondence to Elizabeth J Want or Jeremy K Nicholson.

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Want, E., Masson, P., Michopoulos, F. et al. Global metabolic profiling of animal and human tissues via UPLC-MS. Nat Protoc 8, 17–32 (2013). https://doi.org/10.1038/nprot.2012.135

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