13C metabolic flux analysis is a collective term to indicate a set of methods to experimentally measure in vivo rates through metabolic pathways and reactions. Since reaction rates are not di per se detectable, stable isotopic tracers are used to infer them form the propagation of 13C over time (in dynamic experiments) or from the labeling patterns that emerge when 12C and 13C fragments are merged in metabolism (in end-point measurements). Mass spectrometry is absolutely instrumental to record the labeling patterns in metabolic intermediates and end-products. In real-life problems (see below) the increased problem complexity calls for denser and more accurate 13C data. Hence, 13C metabolic flux analysis strongly depends on improvements in sensitivity, throughput, and coverage of metabolomics.
Different methods exist with heterogeneous demand of inputs and outputs. In our lab, we applied primarily three approaches:
1. Fluxome profiling
Mere statistical analysis of possibly dense 13C-data. Depending on the question, supervised or unsupervised, classification or regression methods can be used.
2. Flux ratio analysis
interpretation of fluxes delivers quantitative information on the
relative (%) fluxes of reactions/pathway converging to a common
intermediate. Flux ratio analysis requires less information and is
amenable to large scale studies, but is difficult to adapt to novel
organisms or environmnents.
3. Isotopomer fitting
When comprehensive and quantitative net flux maps are necessary to e.g. derive cell-wide energy or redox balances, we use the 13CFLUX software by the Wiechert group (FZ Jülich, D). This approach is the most demanding in terms of data, time, and complexity.
Traditional flux analysis methods were developed in the context of metabolic engineering, i.e. for microbial systems and media with single carbon sources. Unfortunately, these systems transfer poorly to more complex conditions
Our current efforts aim at developing new approaches and implement the necessary software to:
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