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Analytical and Bioanalytical Chemistry

, Volume 398, Issue 5, pp 2059–2069 | Cite as

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

  • Ruben t’Kindt
  • Andris Jankevics
  • Richard A. Scheltema
  • Liang Zheng
  • David G. Watson
  • Jean-Claude Dujardin
  • Rainer Breitling
  • Graham H. Coombs
  • Saskia Decuypere
Original Paper

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.
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

Keywords

Metabolomics Leishmania Liquid chromatography–mass spectrometry (LC–MS) HILIC Systems biology 

Notes

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.

Supplementary material

216_2010_4139_MOESM1_ESM.pdf (6.9 mb)
ESM 1 (PDF 7077 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Ruben t’Kindt
    • 1
    • 4
  • Andris Jankevics
    • 2
    • 3
  • Richard A. Scheltema
    • 2
  • Liang Zheng
    • 4
  • David G. Watson
    • 4
  • Jean-Claude Dujardin
    • 1
  • Rainer Breitling
    • 2
    • 3
  • Graham H. Coombs
    • 4
  • Saskia Decuypere
    • 1
  1. 1.Department of Parasitology, Unit of Molecular ParasitologyInstitute of Tropical MedicineAntwerpBelgium
  2. 2.Groningen Bioinformatics Centre, Groningen Biomolecular Sciences and Biotechnology InstituteUniversity of GroningenHarenthe Netherlands
  3. 3.Faculty of Biomedical and Life SciencesUniversity of GlasgowGlasgowUK
  4. 4.Strathclyde Institute of Pharmacy and Biomedical SciencesUniversity of StrathclydeGlasgowUK

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