13C Metabolic Flux Analysis

¹³C metabolic flux analysis (¹³CMFA) 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 the increased problem complexity calls for denser and more accurate ¹³C data. Consequently, ¹³CMFA strongly depends on improvements in sensitivity, throughput, and coverage of metabolomics.

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 measure fluxes in complex situations (ie in higehr cells) and measure fluxes at high-throughputs. 

Targeted ¹³C Metabolic Flux Analysis

Early methods of flux analysis aimed at quantifying possibly many fluxes in central carbon metabolism. This was important in a biotechnological setting, to get a global map of subtrate utilization or derive cellular-wide ATP or redox balances. In contemporary cellular biology, the challenge is different. Quantification of all fluxes is prohibitively difficult. Instead, other approaches such as RNAseq or metabolomics are proficiently used to formulate hypothesis on reactions and pathways that are of particular relevance to drive the metabolic phenotype of interest. For this purpose ¹³CMFA is used to quantify fluxes of very specific reacions or single pathways.

In analogy to othe omics areas, there is a trend towards developing targeted methods that are specialized in a specific task, i.e. offer the best sensitivty while preserving computational and data demand low. We published two such methods for targeted ¹³CMFA: one for non-stationary data (external pagedoicall_made) and one for stationary data (SUMOFLUX link external pagedoicall_made).

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