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

摘要:

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13C全标记的标品。传统制备13C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13C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13C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13C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13C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。

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.