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.
Institute of Biotechnology, ETH Zürich, CH 8093 Zürich, Switzerland.
M
M
Dauner
U
U
Sauer
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