Title
Towards an unbiased metabolic profiling of protozoan parasites: optimisation of a **Leishmania** sampling protocol for HILIC-orbitrap analysis
Author
Faculty/Department
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences . Biomedical Sciences
Publication type
article
Publication
Berlin ,
Subject
Biology
Human medicine
Source (journal)
Analytical and bioanalytical chemistry. - Berlin, 2002, currens
Volume/pages
398(2010) :5 , p. 2059-2069
ISSN
1618-2642
ISI
000283244500027
Carrier
E
Target language
English (eng)
Full text (Publishers DOI)
Affiliation
University of Antwerp
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.
E-info
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