GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing.
Gas chromatography-mass spectrometry (GC-MS) is a rapid method that provides 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 experimental pitfalls are discussed. Second, a suitable interface for the use of mass isotopomer data is presented. For this purpose, a general, matrix-based correction procedure that accounts for naturally occurring isotopes is introduced. Simulated and experimentally determined mass distributions of unlabeled amino acids showed a deviation of less than 3% for 89% of the analyzed fragments. Third, to investigate general properties of GC-MS-based isotopomer balancing, altered flux ratios through glycolysis and pentose phosphate pathway and/or exchange fluxes were simulated. Different fluxes were found to exert specific and significant influence on the mass isotopomer distributions, thus indicating that GC-MS data contain valuable information for metabolic flux analysis. Fourth, comparison of different methods revealed that 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.
Dauner
M
M
Institute of Biotechnology, ETH Zürich, CH 8093 Zürich, Switzerland.
Sauer
U
U
D000596:Amino Acids / Q000032:analysis*; D018533:Biomass; D004926:Escherichia coli / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D008956:Models, Chemical; D012710:Serum Albumin, Bovine / Q000737:chemistry*
D000596:Amino Acids; D012710:Serum Albumin, Bovine