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

Research output: Contribution to journalArticlepeer-review

47 Citations (Scopus)

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 A 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 A 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.

Original languageEnglish
Pages (from-to)2059-2069
Number of pages11
JournalAnalytical and Bioanalytical Chemistry
Volume398
Issue number5
Early online date8 Sept 2010
DOIs
Publication statusPublished - 30 Nov 2010

Keywords

  • metabolomics
  • leishmania
  • liquid chromatography-mass spectrometry (LC-MS)
  • HILIC
  • systems biology
  • LC-MS DATA
  • mass-spectrometry
  • intracellular metabolites
  • visceral leishmaniasis
  • chromatography
  • extraction
  • macrophage
  • capillary
  • reduction
  • algorithm

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