S146: Novel tools for 13C-metabolic flux analysis: tandem mass spectrometry and parallel labeling experiments

¹³C-Metabolic flux analysis (¹³C-MFA) has emerged as a powerful technique for quantifying in vivo metabolism, which is of great importance in fields ranging from metabolic engineering to the study of human metabolic diseases. In this presentation, I will highlight two recent advances in techniques for ¹³C-MFA from our lab: 1) tandem mass spectrometry for measuring isotopomers; and 2) parallel labeling experiments.

1 – Tandem MS is now emerging as a new technique for measuring isotopic-labeling for flux studies. Recently, we have developed computational tools for applying tandem MS for ¹³C-MFA. In addition, we have validated the accuracy and precision of GC-MS/MS measurements and created a library of validated MS/MS fragments. Here, I will demonstrate that using GC-MS/MS we can measure, for the first time, the complete positional isotopomer distributions of metabolites, which is beyond the reach of other measurement technique, and that the precision of estimated fluxes can be improved by >10-fold.

2 – We have also developed a novel parallel labeling strategy for high-resolution ¹³C-MFA, based on optimal selection of tracers. We have shown before that for realistic metabolic networks there will never be a single optimal isotopic tracer for measuring all fluxes in the model. Instead, we developed a new tracer selection strategy based on EMUs that identifies optimal combinations of tracers for parallel experiments, where each tracer is optimal for a specific flux of interest. I will show that combined analysis of parallel labeling experiments provides superior flux results compared to current approaches.