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

Ruben t`Kindt, A Jankevics, RA Scheltema, L Zheng, DG Watson, JC Dujardin, R Breitling, GH Coombs, S Decuypere

    Research output: Contribution to journalA1: Web of Science-article

    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 degrees C, followed by a triplicate washing step in phosphate-buffered saline. The intracellular metabolome of 4 x 10(7) parasites is then extracted in cold chloroform/methanol/water 20/60/20 (v/v/v) for 1 h at 4 degrees 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. Figure 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
    Original languageEnglish
    JournalAnalytical and Bioanalytical Chemistry
    Volume398
    Issue number5
    Pages (from-to)2059-2069
    Number of pages11
    ISSN1618-2642
    DOIs
    Publication statusPublished - 2010

    Keywords

    • B780-tropical-medicine
    • Protozoal diseases
    • Leishmaniasis
    • Leishmania
    • Vectors
    • Sandflies
    • Phlebotomus argentipes
    • Metabolomics
    • Protocols
    • Extraction
    • Liquid
    • Chromatography
    • Mass spectrometry
    • Methodology
    • Laboratory techniques and procedures

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