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Towards an unbiased metabolic profiling of protozoan parasites: optimisation of a Leishmania sampling protocol for HILIC-orbitrap analysis

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An Erratum to this article was published on 11 March 2011

Abstract

Comparative metabolomics of Leishmania species requires the simultaneous identification and quantification of a large number of intracellular metabolites. Here, we describe the optimisation of a comprehensive metabolite extraction protocol for Leishmania parasites and the subsequent optimisation of the analytical approach, consisting of hydrophilic interaction liquid chromatography coupled to LTQ-orbitrap mass spectrometry. The final optimised protocol starts with a rapid quenching of parasite cells to 0 °C, followed by a triplicate washing step in phosphate-buffered saline. The intracellular metabolome of 4 × 107 parasites is then extracted in cold chloroform/methanol/water 20/60/20 (v/v/v) for 1 h at 4 °C, resulting in both cell disruption and comprehensive metabolite dissolution. Our developed metabolomics platform can detect approximately 20% of the predicted Leishmania metabolome in a single experiment in positive and negative ionisation mode.

Final optimized protocol for the study of the intracellular metabolome of Leishmania parasites. Following HILIC-orbitrap analysis of obtained metabolite extracts, 20% of the predicted metabolome is covered, involving metabolites from many different pathways

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Acknowledgements

This work was supported by the GeMInI initiative of the Institute of Tropical Medicine. Ruben t’Kindt was supported by a Research Foundation Flanders Grant for a long stay in Strathclyde. Rainer Breitling was supported by an NWO-Vidi fellowship.

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Authors and Affiliations

  1. Department of Parasitology, Unit of Molecular Parasitology, Institute of Tropical Medicine, 2000, Antwerp, Belgium

    Ruben t’Kindt, Jean-Claude Dujardin & Saskia Decuypere

  2. Groningen Bioinformatics Centre, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN, Haren, the Netherlands

    Andris Jankevics, Richard A. Scheltema & Rainer Breitling

  3. Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK

    Andris Jankevics & Rainer Breitling

  4. Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Taylor Street, Glasgow, G40NR, UK

    Ruben t’Kindt, Liang Zheng, David G. Watson & Graham H. Coombs

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  1. Ruben t’Kindt
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  2. Andris Jankevics
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  3. Richard A. Scheltema
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  4. Liang Zheng
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  5. David G. Watson
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  6. Jean-Claude Dujardin
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  7. Rainer Breitling
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  8. Graham H. Coombs
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  9. Saskia Decuypere
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Corresponding author

Correspondence to Jean-Claude Dujardin.

Additional information

An erratum to this article can be found at http://dx.doi.org/10.1007/s00216-011-4796-7

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t’Kindt, R., Jankevics, A., Scheltema, R.A. et al. Towards an unbiased metabolic profiling of protozoan parasites: optimisation of a Leishmania sampling protocol for HILIC-orbitrap analysis. Anal Bioanal Chem 398, 2059–2069 (2010). https://doi.org/10.1007/s00216-010-4139-0

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