GC-MS-Based Determination of Mass Isotopomer Distributions for ¹³C-Based Metabolic Flux Analysis

Abstract

¹³C-based metabolic flux analysis is currently the most powerful tool to determine intracellular reaction rates in biological systems and valuable, e.g., for the identification of metabolic engineering targets or the elucidation of metabolic pathway activity and regulation. The method exploits that the carbon backbone of metabolites are often manipulated differently by alternative pathways. If the cells are fed with a specifically ¹³C-labeled carbon source, the activity of alternative pathways determines the incorporation of the stable isotopes into metabolites and biomass constituents resulting in pathway-specific labeling patterns. In conventional ¹³C-MFA, cells in metabolic (pseudo-)steady state, i.e., during exponential growth or during steady-state continuous cultivation, are fed with a ¹³C-labeled carbon source and harvested when the metabolic intermediates or biomass constituents have reached an isotopic labeling steady state. The method is today applied on all types of cells, from microbial to plant to mammalian cells or whole organs, and on diverse carbon sources. State-of-the-art ¹³C-based metabolic flux analysis most often applies mass spectrometry for the determination of ¹³C-labeling patterns in intracellular metabolites. Integration of the ¹³C-enrichment data with biochemical reaction networks and metabolic modeling allows then the calculation of intracellular fluxes.

In this chapter, we give a step-by-step protocol for the setup and validation of gas chromatography-mass spectrometry-based analysis of ¹³C-mass isotopomer distributions of proteinogenic amino acids.