[Preparation of isotope dilution mass spectrometry standards based on glucose pulse]

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.