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

作者：Cipollina Chiara; ten Pierick Angela; Canelas Andre B; Seifar Reza M; van Maris Antonius J A; van Dam Jan C^*; Heijnen Joseph J

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2009, 877(27): 3231-3236.

DOI：10.1016/j.jchromb.2009.07.019

• Methoxime-trimethylsilyl (MOX-TMS) derivatives
• GC-MS
• Isotope dilution mass spectrometry
• Metabolomics
• Pentose phosphate pathway
• Saccharomyces cerevisiae

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 (13)C-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.

• 出版日期2009-10-1