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Tissue disruption and extraction methods for metabolic profiling of an invertebrate sentinel species

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Abstract

Metabolic profiling of tissues needs special attention, because the compartmentalization of cellular constituents will be abolished by sample homogenization. This loss of partitioning leads to protein and metabolite instability in extracts, and therefore metabolite extraction protocols need to ensure very rapid inactivation of macromolecules as well as solubilization of metabolites. There are many published methods for tissue metabolome analysis, but no universally accepted standard, and a lack of measurable quality benchmarks. We developed a protocol for efficient tissue disruption and metabolite extraction of the earthworm Lumbricus rubellus guided by prior biological knowledge as well as metrics based on the data. In particular, we identified an unusual degree of instability of L. rubellus tissue extracts, and evaluated different approaches such as heating and filtration to counteract this. Finally, we evaluated four different solvent systems for comprehensive metabolite extraction using three analytical platforms (1H NMR spectroscopy, GC–MS, and direct-infusion FT-ICR-MS), and also compared bead-beating and cryogenic milling for tissue disruption. Initially we ranked methods by common analytical criteria (e.g. numbers and total intensity of detected peaks) in order to compare protocols. These approaches to assess protocol suitability proved to be inadequate to judge earthworm tissue extraction methods because of sample instability. Existing tissue extraction protocols should not be assumed to be automatically applicable to novel species.

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Acknowledgments

We thank Olaf Beckonert (Imperial College London) for assistance with NMR spectroscopy, Volker Behrends (Imperial College London) for assistance with data processing, Mark Viant and Ulf Sommer (University of Birmingham) for discussion of MS data, and David Spurgeon and Claus Svendsen (CEH Wallingford) for supplying L. rubellus worms and scientific discussion. This work was supported by the UK Natural Environment Research Council (NERC), grant number NE/H009973/1, and in part supported by the NERC Biomolecular Analysis Facility at the University of Birmingham (R8-H10-61) with the FT-ICR MS analysis conducted by Ulf Sommer and Mark Viant. This instrument was obtained through the Birmingham Science City Translational Medicine: Experimental Medicine Network of Excellence project, with support from Advantage West Midlands.

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  1. Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, SW7 2AZ, UK

    Manuel Liebeke & Jacob G. Bundy

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  1. Manuel Liebeke
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Correspondence to Jacob G. Bundy.

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11306_2011_377_MOESM1_ESM.pdf

Table S1 listing methods used for tissue extraction in other earthworm metabolomics studies. Supporting methods information S2. Figures S3 – S7. (PDF 699 kb)

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Liebeke, M., Bundy, J.G. Tissue disruption and extraction methods for metabolic profiling of an invertebrate sentinel species. Metabolomics 8, 819–830 (2012). https://doi.org/10.1007/s11306-011-0377-1

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