Preparation of isotope dilution mass spectrometry standards based on glucose pulse.

Wei Shu; Chao LI; Xiaoyun Liu; Jianye Xia; Yingping Zhuang

Return

Preparation of isotope dilution mass spectrometry standards based on glucose pulse.

Author: Wei SHU ¹ ; Chao LI ¹ ; Xiaoyun LIU ¹ ; Jianye XIA ¹ ; Yingping ZHUANG ¹
Author Information

1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Publication Type:Journal Article
Keywords: GC-MS; IDMS standard; LC-MS; Pichia pastoris G/DSEL; fast sampling; substrates pulse
From: Chinese Journal of Biotechnology 2017;33(11):1869-1876
CountryChina
Language:Chinese
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.