GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing

Gas chromatography-mass spectrometry (GC-MS) is a rapid method that provide s rich information on isotopomer distributions for metabolic flux analysis. First, we established a fast and reliable experimental protocol for GC-MS analysis of amino acids from total biomass hydrolyzates, and common experim ental pitfalls are discussed. Second, a suitable interface for the use of m ass isotopomer data is presented. For this purpose, a general, matrix-based correction procedure that accounts for naturally occurring isotopes is int roduced. Simulated and experimentally determined mass distributions of unla beled amino acids showed a deviation of less than 3% for 89% of the analyze d fragments. Third, to investigate general properties of GC-MS-based isotop omer balancing, altered flux ratios through glycolysis and pentose phosphat e pathway and/or exchange fluxes were simulated. Different fluxes were foun d to exert specific and significant influence on the mass isotopomer distri butions, thus indicating that GC-MS data contain valuable information for m etabolic flux analysis, Fourth, comparison of different methods revealed th at GC-MS analysis provides the largest number of independent constraints on amino acid isotopomer abundance, followed by correlation spectroscopy and fractional enrichment analysis by nuclear magnetic resonance.