Sample preparation for intracellular metabolite extraction in T. b. brucei 427

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SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".

The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.

Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...

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 ...

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 ...

In addition to the highly targeted quantification of metabolites already known to play major roles in oxidative stress, to provide data directly compatible with current models, we will also take an untargeted metabolomics approach. This will enable us to identify other areas of the metabolome influenced by, or influencing, oxidative stress and will allow us to compare changes in each of the stress-inducing stimuli. We have recently pioneered untargeted metabolite profiling of T. brucei using ...

We have already demonstrated that the key metabolites of polyamine biosynthesis (arginine, ornithine, putrescine and spermidine) can be identified using HILIC chromatography coupled to the Orbitrap mass spectrometer, as can glycine, glutamate and cysteine used in glutathione biosynthesis, glutathionyl spermidine and trypanothione itself. Furthermore the key metabolites of the methionine cycle (methionine, S-adenosyl methionine, decarboxylated S-adenosyl methionine, methylthioadenosine) can all ...

Intracellular 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.

Metabolite profiling on T. brucei exposed to methylene blue has been carried out using LC-MS to investigate metabolic changes caused by oxidative stress

26 intracellular metabolites (amino acids, polyamines, TCA intermediates) in T. brucei exposed to methylene blue have been absolutely quantified using isotope ratio based MS technique.

26 intracellular metabolites (amino acids, polyamines, TCA intermediates) in T. brucei under pH stress (pH8.7) have been absolutely quantified using isotope ratio based MS technique.