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Springer (part of Springer Nature), Analytical and Bioanalytical Chemistry, 5(398), p. 2059-2069

DOI: 10.1007/s00216-010-4139-0

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

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This paper is available in a repository.

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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 ?? 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. ?? 2010 {Springer-Verlag.}