Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 398, Issue 5, Pages 2059-2069Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-010-4139-0
Keywords
Metabolomics; Leishmania; Liquid chromatography-mass spectrometry (LC-MS); HILIC; Systems biology
Funding
- GeMInI initiative of the Institute of Tropical Medicine
- Research Foundation Flanders
- NWO
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available