LC-MS-based absolute metabolite quantification: application to metabolic flux measurement in trypanosomes.
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-labelledE.coliextracts as internal standards. Known amounts of labelledE. coliextract 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.
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA UK ; Centre for Analytical Bioscience, School of Pharmacy, University of Nottingham, University Park, Nottingham, NG7 2RD UK.
Dong-Hyun
DH
Kim
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA UK.
Fiona
F
Achcar
Manchester Centre of Synthetic Biology for Fine and Speciality Chemicals, Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, Manchester, M1 7DN UK.
Rainer
R
Breitling
Glasgow Polyomics, Wolfson Wohl Cancer Research Centre, College of Medical Veterinary & Life Sciences, University of Glasgow, Glasgow, G61 1QH UK.
Karl E
KE
Burgess
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA UK ; Glasgow Polyomics, Wolfson Wohl Cancer Research Centre, College of Medical Veterinary & Life Sciences, University of Glasgow, Glasgow, G61 1QH UK.
Michael P
MP
Barrett
13C-labelled E. coli; Absolute metabolite quantification; LC–MS; Metabolic flux; Trypanosome