Preparation of Isotope Dilution Mass Spectrometry standards based on glucose pulse

Abstract:

Isotope Dilution Mass Spectrometry (IDMS) is the most accurate method for high-throughput detection of intracellular metabolite concentrations, and the key is getting the corresponding fully uniformly(U) 13C-labeled metabolites to be measured. The conventional procedure for getting fully U 13C-labeled metabolites is through batch cultivation, but intracellular metabolites concentrations by this method are generally low. By applying U 13C-labeled glucose pulse, combined with fast sampling and quenching, mixture of fully U 13C-labeled intracellular metabolites was successfully extracted with higher concentration from Pichia pastoris G/DSEL fed with fully U 13C-labeled glucose as only carbon source. Quantitative results from liquid chromatography tandem mass spectrometry (LC-MS) and gas chromatography tandem mass spectrometry (GC-MS) show that concentrations of organic acids, sugar phosphates, amino acids and nucleotides were 2–10 folds higher than those without glucose pulse. Therefore, the glucose pulse method can efficiently improve the usage of fully U 13C-labeled glucose converting to 13C-labeled metabolites, and achieve the detection of intracellular metabolites with lower concentrate than the instrument detection limit.