A comprehensive method for the quantification of the non-oxidative pentose phosphate pathway intermediates in Saccharomyces cerevisiae by GC-IDMS.
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, 10m, 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.
Cipollina
Chiara
C
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
ten Pierick
Angela
A
Canelas
André B
AB
Seifar
Reza M
RM
van Maris
Antonius J A
AJ
van Dam
Jan C
JC
Heijnen
Joseph J
JJ
D002247:Carbon Isotopes / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D005986:Glyceraldehyde 3-Phosphate / Q000032:analysis; D007553:Isotope Labeling; D055432:Metabolomics / Q000379:methods; D010091:Oximes / Q000737:chemistry; D010427:Pentose Phosphate Pathway; D010428:Pentosephosphates / Q000032:analysis*; D015203:Reproducibility of Results; D012274:Ribulosephosphates / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013403:Sugar Phosphates / Q000032:analysis; D014297:Trimethylsilyl Compounds / Q000737:chemistry
D002247:Carbon Isotopes; D010091:Oximes; D010428:Pentosephosphates; D012274:Ribulosephosphates; D013403:Sugar Phosphates; D014297:Trimethylsilyl Compounds; D005986:Glyceraldehyde 3-Phosphate; C031524:ribulose 5-phosphate; C026959:erythrose 4-phosphate; C031625:xylulose-5-phosphate; C005514:N,N'-monomethylenebis(pyridiniumaldoxime)