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

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 study, we tried to make it possible by combining ultra-high performance liquid chromatography-triple quadrupole mass spectrometry method 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 chromatographic 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 was established. The results showed that this method 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.

13C internal standards which extracted from Pichia Pastoris cultured using U13C glucose as sole carbon source were added into samples. Mixed samples were detected using LC-MS/MS. The metabolites and isotope analogues were co-eluted but could be distinguished by mass spectrometry, and we could quantify the metabolites more accurately.Figure optionsDownload as PowerPoint slide