Metabolomics

, Volume 8, Issue 5, pp 819–830

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

Original Article

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.

Keywords

Metabolomics NMR Lumbricus rubellus Tissue extraction Instability GC–MS Direct infusion mass spectrometry Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) 

Supplementary material

11306_2011_377_MOESM1_ESM.pdf (700 kb)
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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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Biomolecular Medicine, Department of Surgery and Cancer, Faculty of MedicineImperial College LondonLondonUK

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