Sample preparation procedure for footprint analysis on T. b. brucei 427 using LC/MS

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The SilicoTryp project aims at the creation of a “Silicon Trypanosome”, a comprehensive, experiment-based, multi-scale mathematical model of trypanosome physiology.
Trypanosomes are blood-stream parasites transmitted by tsetse flies; they cause African sleeping sickness in humans and livestock. Currently available drugs have severe side effects, and the parasites are rapidly developing resistance.
In this project, we collect a wide range of new experimental data on the parasite in its various
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The aims of this investigation is to quantify metabolites associated with pathways involved in stress responses for parameterising models of oxidative stress metabolism; the measurement of metabolic fluxes of metabolites of interest with intracellular concentrations

Flux will be measured using the metabolomics platforms based on absolute quantification method (isotope ratio based MS technique) by LC-MS, using heavy-isotope labelled precursors of the metabolites of interest. For example, 15N labelled cysteine, glycine and glutamate will be used to determine rates of synthesis of glutathione. 15N-labelled methionine to measure S-adenosyl methionine (and its decarboxylated form, as well as methionine cycle intermediates). 15N labelled arginine is used as precursor
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Extracellular metabolites in T. brucei at different stage of cell growth have been quantified absolutely by isotope ratio based MS technique using uniformly 13C-labelled E. coli extract. This is the case study for method development of absolute quantification for metabolic flux analysis.