~(13)C辅助的超高效液相色谱-三重四极杆质谱联用方法精确分析毕赤酵母胞内代谢物浓度
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摘要
毕赤酵母作为一种高效的外源蛋白表达平台,其蛋白表达水平与胞内代谢物浓度紧密相关。但胞内代谢物种类多、物化性质差异大、浓度低、周转快,对其绝对浓度的精确检测一直难于实现。本研究将超高效液相色谱-三重四极杆质谱联用分析方法与13C同位素标记技术相结合,探索解决该难题的方法。首先,优化了超高效液相色谱的操作条件,利用3种色谱柱实现了64种常见中间代谢物的分离;对三重四极杆质谱仪的检测离子对和碰撞电压等操作条件进行优化,找到了对各种物质具有专一性的检测离子对。然后,利用全标记13C标记底物培养细胞,收集胞内的全标记代谢物用作定量内标物,建立了53种中间代谢物的标准曲线。实验结果表明,本方法不但精确性高,标准曲线相关系数达到0.99以上,而且重现性好,受实验条件和仪器操作条件的影响很小。将本方法应用于毕赤酵母胞内代谢物浓度的绝对定量分析,成功获得了胞内各种代谢物的浓度水平,为后续深入研究毕赤酵母代谢调控机理,实现外源蛋白的高效生产奠定了基础。
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 ~(13)C 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 U~(13)C 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.
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 ~(13)C 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 U~(13)C 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.
引文
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14 LI Min-Chao,HUANG Ming-Zhi,LIU Yu-Wei,CHU Ju,ZHUANG Ying-Ping,ZHANG Si-Liang.Chinese J.Anal.Chem.,2014,42(10):1408-1403李敏超,黄明志,刘玉伟,储炬,庄英萍,张嗣良.分析化学,2014,42(10):1408-1403
15 JordàJ,Suarez C,Carnicer M,ten Pierick A,Heijnen J J,Gulik W,Ferrer P,Albiol J,Wahl A.BMC Syst.Biol.,2013,7(1):1-16
16 JordàJ,Jouhten P,Cámara E,Maaheimo H,Albiol J,Ferrer P.Microb.Cell Fact.,2012,11:57
2 Ogawa T,Washio J,Takahashi T,Echigo S,Takahashi N.Oral Surg.Oral.Med.Oral.Pathol.Oral.Radiol.,2014,118(2):218-225
3 Xia M L,Huang D,Li S S,Wen J P,Jia X Q,Chen Y L.Biotechnol.Bioeng.,2013,110(10):2717-2730
4 Wasylenko T M,Stephanopoulos G.Biotechnol.Bioeng,2014,112(3):470-483
5 Buchholz A,Takors R,Wandrey C.Anal.Biochem.,2001,295(2):129-137
6 van Dam J C,Eman M,Frank J,Lange H C,van Dedem G W K,Heijnen S J.Anal.Chim.Acta,2002,460(2):209-218
7 Wu L,Mashego M R,van Dam J C,Proell A M,Vinke J L,Ras C,van Winden W A,van Gulik W M,Heijnen J J.Anal.Biochem.,2005,336(2):164-171
8 Bennett B D,Yuan J,Kimball E H,Rabinowitz J D.Nature Protocols,2008,3(8):1299-1311
9 Hellerstein M K,Neese R A.Am.J.Physiol,1999,276(6):1146-1170
10 Mc Closkey D,Gangoiti G A,King Z A,Naviaux R K,Barshop B A,Palsson B O,Feist A M.Biotechnol.Bioeng.,2014,111(4):803-815
11 Carnicer M,Canelas A.B,Pierick A,Zeng Z,Dam J,Albiol J,Ferrer P,Heijnen J J,Gulik W.Metabolomics,2012,8(2):284-298
12 Tredwell G D,Edwards-Jones B,Leak D J,Bundy J G.PLo S One,2011,6(1):e16286
13 JordàJ,Rojas H C,Carnicer M,Wahl A,Ferrer P,Albiol J.Metabolites,2014,4(2):281-299
14 LI Min-Chao,HUANG Ming-Zhi,LIU Yu-Wei,CHU Ju,ZHUANG Ying-Ping,ZHANG Si-Liang.Chinese J.Anal.Chem.,2014,42(10):1408-1403李敏超,黄明志,刘玉伟,储炬,庄英萍,张嗣良.分析化学,2014,42(10):1408-1403
15 JordàJ,Suarez C,Carnicer M,ten Pierick A,Heijnen J J,Gulik W,Ferrer P,Albiol J,Wahl A.BMC Syst.Biol.,2013,7(1):1-16
16 JordàJ,Jouhten P,Cámara E,Maaheimo H,Albiol J,Ferrer P.Microb.Cell Fact.,2012,11:57