LC–MS-based absolute metabolite quantification: application to metabolic flux measurement in trypanosomes

Kim, D.-H., Achcar, F., Breitling, R. , Burgess, K. E. and Barrett, M. P. (2015) LC–MS-based absolute metabolite quantification: application to metabolic flux measurement in trypanosomes. Metabolomics, 11(6), pp. 1721-1732. (doi:10.1007/s11306-015-0827-2) (PMID:26491423) (PMCID:PMC4605981)

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Abstract

Human African trypanosomiasis is a neglected tropical disease caused by the protozoan parasite, Trypanosoma brucei. In the mammalian bloodstream, the trypanosome’s metabolism differs significantly from that of its host. For example, the parasite relies exclusively on glycolysis for energy source. Recently, computational and mathematical models of trypanosome metabolism have been generated to assist in understanding the parasite metabolism with the aim of facilitating drug development. Optimisation of these models requires quantitative information, including metabolite concentrations and/or metabolic fluxes that have been hitherto unavailable on a large scale. Here, we have implemented an LC–MS-based method that allows large scale quantification of metabolite levels by using U-13C-labelled E. coli extracts as internal standards. Known amounts of labelled E. coli extract were added into the parasite samples, as well as calibration standards, and used to obtain calibration curves enabling us to convert intensities into concentrations. This method allowed us to reliably quantify the changes of 43 intracellular metabolites and 32 extracellular metabolites in the medium over time. Based on the absolute quantification, we were able to compute consumption and production fluxes. These quantitative data can now be used to optimise computational models of parasite metabolism.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Achcar, Dr Fiona and Breitling, Professor Rainer and Burgess, Dr Karl and Barrett, Professor Michael
Authors: Kim, D.-H., Achcar, F., Breitling, R., Burgess, K. E., and Barrett, M. P.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Metabolomics
Publisher:Springer US
ISSN:1573-3882
ISSN (Online):1573-3890
Copyright Holders:Copyright © 2015 The Authors
First Published:First published in Metabolomics 11(6):1721-1732
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
522081The silicon trypanosome (SilicoTryp)Michael BarrettBiotechnology and Biological Sciences Research Council (BBSRC)BB/I004599/1III - PARASITOLOGY