Towards an unbiased metabolic profiling of protozoan parasites: optimisation of a Leishmania sampling protocol for HILIC-orbitrap analysis.
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 × 10(7) 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.
t'Kindt
Ruben
R
Department of Parasitology, Unit of Molecular Parasitology, Institute of Tropical Medicine, 2000 Antwerp, Belgium.
Jankevics
Andris
A
Scheltema
Richard A
RA
Zheng
Liang
L
Watson
David G
DG
Dujardin
Jean-Claude
JC
Breitling
Rainer
R
Coombs
Graham H
GH
Decuypere
Saskia
S
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D007891:Leishmania / Q000737:chemistry* / Q000254:growth & development; D055442:Metabolome