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

t’Kindt, R., Jankevics, A., Scheltema, R.A., Zheng, L., Watson, D.G., Dujardin, J.C., Breitling, R. , Coombs, G.H. and Decuypere, S. (2010) Towards an unbiased metabolic profiling of protozoan parasites: optimisation of a Leishmania sampling protocol for HILIC-orbitrap analysis. Analytical and Bioanalytical Chemistry, 398(5), pp. 2059-2069. (doi: 10.1007/s00216-010-4139-0)

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 A 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 A 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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Coombs, Professor Graham and Breitling, Professor Rainer
• Authors: t’Kindt, R., Jankevics, A., Scheltema, R.A., Zheng, L., Watson, D.G., Dujardin, J.C., Breitling, R., Coombs, G.H., and Decuypere, S.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: Analytical and Bioanalytical Chemistry
• ISSN: 1618-2642