Analysis of glycolytic intermediates in Saccharomyces cerevisiae using anion exchange chromatography and electrospray ionization with tandem mass spectrometric detection

The purpose of this study is to selectively and quantitatively analyze several glycolytic intermediates in cells of Saccharomyces cerevisiae using high-performance anion exchange chromatography (HPAEC) coupled to electrospray ionization tandem mass spectrometry for the analysis. A sodium hydroxide gradient is used to separate the glycolytic compounds and after the column sodium hydroxide is reduced by proton exchange with a membrane device prior to introduction to the mass spectrometer. The detection limits for 10 mul samples are down to the 0.4-5 pmol range. This corresponds for the intracellular metabolites to a range of 2-20 nmol per gram biomass dry weight (DW), Standard addition did reveal some influence of the sample matrix on the measured concentrations. Separation and analysis is hardly affected by the high sulfate and phosphate concentrations (1 mM) in the fermentation medium and by the intracellular matrix. Validation of the glucose-6-phosphosphate LC-MS-MS analysis results with enzymatic analysis showed an excellent agreement between the two methods. The suitability of the method was clearly shown by analyzing a series of steady state S. cerevisiae samples from a carbon limited aerobic chemostat culture. (C) 2002 Elsevier Science B.V. All rights reserved.