基于葡萄糖脉冲的同位素稀释质谱检测法标品制备

Preparation of isotope dilution mass spectrometry standards based on glucose pulse

同位素稀释质谱检测法(IDMS)是目前进行胞内代谢物浓度高通量检测精度最高的一种方法,这个方法的关键就是制备待检测代谢物对应的^(13)C全标记的标品。传统制备^(13)C全标记标品的方法是采用批培养的模式获取,但该方法所制备的胞内代谢物浓度通常较低。通过以^(13)C全位标记葡萄糖作为唯一碳源培养毕赤酵母G/DSEL菌种,采用^(13)C全位标记葡萄糖脉冲刺激法,结合快速取样淬灭方法的新方法,成功制备了带^(13)C标记标品并提高了其浓度。经液质联用(LC-MS)与气质联用(GC-MS)结果分析,与传统制备方法相比,胞内大部分有机酸、磷酸糖、氨基酸和核苷酸类物质的浓度实现了约2–10倍的提高。因此底物脉冲法可以有效提高^(13)C全标葡萄糖的单位利用率,并能实现对胞内部分含量低于仪器检测限的代谢物的检测。

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 metabolmtes 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.