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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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| Abstract |
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| 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. | | |
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English
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Analytical and bioanalytical chemistry. - Berlin, 2002, currens | |
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Berlin : 2010
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1618-2642
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398:5(2010), p. 2059-2069
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000283244500027
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