Citation: GUO Meng-Lei, LIU Xiao-Yun, HUANG Ming-Zhi, LI Min-Chao, CHU Ju, ZHUANG Ying-Ping, ZHANG Si-Liang. ¹³C-assisted Ultra-high Performance Liquid Chromatography Triple Quadrupole Mass Spectrometry Method for Precise Determination of Intracellular Metabolites in Pichia pastoris[J]. Chinese Journal of Analytical Chemistry, ;2016, 44(2): 232-240. doi: 10.11895/j.issn.0253-3820.150635 shu

¹³C-assisted Ultra-high Performance Liquid Chromatography Triple Quadrupole Mass Spectrometry Method for Precise Determination of Intracellular Metabolites in Pichia pastoris

1.
National Engineering Research Center for Biotechnology (Shanghai), East China University of Science and Technology, Shanghai 200237, China

Corresponding author: HUANG Ming-Zhi,
Received Date: 10 August 2015
Available Online: 29 October 2015

The yeast Pichia pastoris is an effective host for recombinant protein production and the recombinant protein production level is tightly related to the concentrations of intracellular metabolites. The intracellular metabolites have the features of wide range of types, distinct variation in physical and chemical properties, rapid turning-over and low concentration, so it is difficult to quantify their concentrations precisely. In this experiment, we tried to make it possible by combining ultra-high performance liquid chromatography (UPLC)-triple quadrupole mass spectrometry and ¹³C isotope labeling techniques. 64 metabolites including organic acids, sugar phosphate, nucleoside substance, amino acid were successfully separated by UPLC with three kinds of chromatography column. The appropriate and unique ion pairs and collision voltages were found after the mass spectrometry condition optimization. By using the U¹³C metabolites as internal standards collected from the cells growing on U¹³C-glucose as sole carbon source, the standard curves of 53 metabolites were established. The results showed that the method presented here had a high accuracy. The correlation coefficients were above 0.99. The method also had a good reproducibility, and the influence of experimental and equipment operating condition was very small. Reliable and accurate determination of the concentrations of intracellular metabolites in Pichia pastoris was obtained. The works lays the foundation for comprehensive regulation mechanism research and efficient recombinant protein production in Pichia pastoris.
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通讯作者: 陈斌, bchen63@163.com

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沈阳化工大学材料科学与工程学院沈阳 110142

13C-assisted Ultra-high Performance Liquid Chromatography Triple Quadrupole Mass Spectrometry Method for Precise Determination of Intracellular Metabolites in Pichia pastoris

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通讯作者: 陈斌, bchen63@163.com

¹³C-assisted Ultra-high Performance Liquid Chromatography Triple Quadrupole Mass Spectrometry Method for Precise Determination of Intracellular Metabolites in Pichia pastoris