|
Title
|
|
|
|
Towards an unbiased metabolic profiling of protozoan parasites: optimisation of a **Leishmania** sampling protocol for HILIC-orbitrap analysis
|
|
|
Author
|
|
|
|
|
|
|
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 × 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. |
|
|
|
Language
|
|
|
|
English
|
|
|
Source (journal)
|
|
|
|
Analytical and bioanalytical chemistry / Gesellschaft Deutscher Chemiker. - Heidelberg, 2002, currens
|
|
|
Publication
|
|
|
|
Heidelberg
:
Springer
,
2010
|
|
|
ISSN
|
|
|
|
1618-2642
[print]
1618-2650
[online]
|
|
|
DOI
|
|
|
|
10.1007/S00216-010-4139-0
|
|
|
Volume/pages
|
|
|
|
398
:5
(2010)
, p. 2059-2069
|
|
|
ISI
|
|
|
|
000283244500027
|
|
|
Full text (Publisher's DOI)
|
|
|
|
|
|