13C Isotopomer Analysis of Glutamate by Tandem Mass Spectrometry

Abstract

Tandem mass spectrometry allows a compound to be isolated from the rest of the sample and dissociated into smaller fragments. We show here that fragmentation of glutamate mass isotopomers yields additional mass spectral data that significantly improve the analysis of metabolic fluxes compared to full-scan mass spectrometry. In order to validate the technique, tandem and full-scan mass spectrometry were used along with 13C NMR to analyze glutamate from rat hearts perfused with three substrate mixtures (5 mM glucose plus 5 mM [2-13C]acetate, 5 mM [1-13C]glucose plus 5 U/L insulin, and 5 mM glucose plus 1 mM [3-13C]pyruvate). Analysis by tandem mass spectrometry showed that the enriched substrate contributed 98 ± 2, 53 ± 2, and 84 ± 7%, respectively, of acetyl-coenzyme A while the rate of anaplerotic substrate entry was 7 ± 3, 25 ± 8, and 16 ± 8%. Similar results were obtained with 13C NMR data, while values from full-scan data had higher error. We believe that this is the first use of tandem mass spectrometry to determine pathway flux using 13C-enriched substrates. Although analysis of the citric acid cycle by NMR is simpler (and more intuitive), tandem mass spectrometry has the potential to combine high sensitivity with the high information yield previously available only by NMR.