A comprehensive method for the quantification of the non-oxidative pentose phosphate pathway intermediates in Saccharomyces cerevisiae by GC–IDMS

Abstract

A gas chromatography isotope dilution mass spectrometry (GC–IDMS) method was developed for the quantification of the metabolites of the non-oxidative part of pentose phosphate pathway (PPP). A mid-polar GC column (Zebron ZB-AAA, 10 m, film composition 50% phenyl 50% dimethyl polysiloxane) was used for the chromatographic separation of the intermediates. The optimized GC–MS procedure resulted in improved separation performances and higher sensitivities compared to previous methods. Furthermore, the use of 13C-labeled cell extracts as internal standards improved the data quality and eliminated the need to perform a recovery check for each metabolite. The applicability of the new method was demonstrated by analyzing intracellular metabolite levels in samples derived from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae at steady state as well as following a short-term glucose pulse. The major achievements of the proposed quantitative method are the independent quantification of the epimers ribulose-5-phosphate and xylulose-5-posphate and the measurement of compounds present at very low concentrations in biological samples such as erythrose-4-phosphate and glyceraldehyde-3-phosphate.