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 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 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. Figure Final optimized protocol for the study of the intracellular metabolome of Leishmania parasites. Following HILIC-orbitrap analysis of obtained metabolite extracts, 20% of the predicted metabolome is covered, involving metabolites from many different pathways