Mass spectrometry for metabolic flux analysis

Mass spectrometry in combination with tracer experiments based on C-13 subs trates can serve as a powerful tool for the modeling and analysis of intrac ellular fluxes and the investigation of biochemical networks. The theoretic al background for the application of mass spectrometry to metabolic flux an alysis is discussed. Mass spectrometry methods are especially useful to det ermine mass distribution of metabolites. Additional information gained from fragmentation of metabolites, e.g., by electron impact ionization, allows further localization of labeling positions, up to complete resolution of is otopomer pools. To effectively handle mass distributions in simulation expe riments, a matrix based general methodology is formulated. The natural isot ope distribution of carbon, oxygen, hydrogen and nitrogen in the target met abolites is considered by introduction of correction matrices. It is shown by simulation results for the central carbon metabolism that neglecting nat ural isotope distributions causes significant errors in intracellular flux distributions. By varying relative fluxes into pentosephosphate pathway and pyruvate carboxylation reaction, marked changes in the mass distributions of metabolites result, which are illustrated for pyruvate, oxaloacetate, an d alpha-ketoglutarate. In addition mass distributions of metabolites are si gnificantly influenced over a broad range by the degree of reversibility of transaldolase and transketolase reactions in the pentosephosphate pathway. The mass distribution of metabolites is very sensitive towards intracellul ar flux patterns and can be measured with high accuracy by routine mass spe ctrometry methods. (C) 1999 John Wiley & Sons, Inc.