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.
1. [1]
  1 Fan H, Li S P, Xiang J J, Lai C M, Yang F Q, Gaoa J L, Wang Y T. Anal. Chim. Acta, 2006, 567(2): 218-228
2. [2]
  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. [3]
  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. [4]
  4 Wasylenko T M, Stephanopoulos G. Biotechnol. Bioeng, 2014, 112(3): 470-483
5. [5]
  5 Buchholz A, Takors R, Wandrey C. Anal. Biochem., 2001, 295(2): 129-137
6. [6]
  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. [7]
  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. [8]
  8 Bennett B D, Yuan J, Kimball E H, Rabinowitz J D. Nature Protocols, 2008, 3(8): 1299-1311
9. [9]
  9 Hellerstein M K, Neese R A. Am. J. Physiol, 1999, 276(6): 1146-1170
10. [10]
  10 McCloskey 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. [11]
  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. [12]
  12 Tredwell G D, Edwards-Jones B, Leak D J, Bundy J G. PLoS One, 2011, 6(1): e16286
13. [13]
  13 Jordà J, Rojas H C, Carnicer M, Wahl A, Ferrer P, Albiol J. Metabolites, 2014, 4(2): 281-299
14. [14]
  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. [15]
  15 Jordà J, 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. [16]
  16 Jordà J, Jouhten P, Cámara E, Maaheimo H, Albiol J, Ferrer P. Microb. Cell Fact., 2012, 11: 57
1. [1]
  Yanhui Zhong , Ran Wang , Zian Lin . Analysis of Halogenated Quinone Compounds in Environmental Water by Dispersive Solid-Phase Extraction with Liquid Chromatography-Triple Quadrupole Mass Spectrometry. University Chemistry, 2024, 39(11): 296-303. doi: 10.12461/PKU.DXHX202402017
2. [2]
  Yaling Chen . Basic Theory and Competitive Exam Analysis of Dynamic Isotope Effect. University Chemistry, 2024, 39(8): 403-410. doi: 10.3866/PKU.DXHX202311093
3. [3]
  Mingyang Men , Jinghua Wu , Gaozhan Liu , Jing Zhang , Nini Zhang , Xiayin Yao . 液相法制备硫化物固体电解质及其在全固态锂电池中的应用. Acta Physico-Chimica Sinica, 2025, 41(1): 2309019-. doi: 10.3866/PKU.WHXB202309019
4. [4]
  Cunling Ye , Xitong Zhao , Hongfang Wang , Zhike Wang . A Formula for the Calculation of Complex Concentrations Arising from Side Reactions and Its Applications. University Chemistry, 2024, 39(4): 382-386. doi: 10.3866/PKU.DXHX202310043
5. [5]
  Junjie Zhang , Yue Wang , Qiuhan Wu , Ruquan Shen , Han Liu , Xinhua Duan . Preparation and Selective Separation of Lightweight Magnetic Molecularly Imprinted Polymers for Trace Tetracycline Detection in Milk. University Chemistry, 2024, 39(5): 251-257. doi: 10.3866/PKU.DXHX202311084
6. [6]
  Yan LIU , Jiaxin GUO , Song YANG , Shixian XU , Yanyan YANG , Zhongliang YU , Xiaogang HAO . Exclusionary recovery of phosphate anions with low concentration from wastewater using a CoNi-layered double hydroxide/graphene electronically controlled separation film. Chinese Journal of Inorganic Chemistry, 2024, 40(9): 1775-1783. doi: 10.11862/CJIC.20240043
7. [7]
  Changqing MIAO , Fengjiao CHEN , Wenyu LI , Shujie WEI , Yuqing YAO , Keyi WANG , Ni WANG , Xiaoyan XIN , Ming FANG . Crystal structures, DNA action, and antibacterial activities of three tetranuclear lanthanide-based complexes. Chinese Journal of Inorganic Chemistry, 2024, 40(12): 2455-2465. doi: 10.11862/CJIC.20240192
8. [8]
  Zian Lin , Yingxue Jin . Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) for Disease Marker Screening and Identification: A Comprehensive Experiment Teaching Reform in Instrumental Analysis. University Chemistry, 2024, 39(11): 327-334. doi: 10.12461/PKU.DXHX202403066
9. [9]
  Zunxiang Zeng , Yuling Hu , Yufei Hu , Hua Xiao . Analysis of Plant Essential Oils by Supercritical CO₂Extraction with Gas Chromatography-Mass Spectrometry: An Instrumental Analysis Comprehensive Experiment Teaching Reform. University Chemistry, 2024, 39(3): 274-282. doi: 10.3866/PKU.DXHX202309069
10. [10]
  Hao Wu , Zhen Liu , Dachang Bai . ¹H NMR Spectrum of Amide Compounds. University Chemistry, 2024, 39(3): 231-238. doi: 10.3866/PKU.DXHX202309020
11. [11]
  Kexin Dong , Chuqi Shen , Ruyu Yan , Yanping Liu , Chunqiang Zhuang , Shijie Li . Integration of Plasmonic Effect and S-Scheme Heterojunction into Ag/Ag₃PO₄/C₃N₅ Photocatalyst for Boosted Photocatalytic Levofloxacin Degradation. Acta Physico-Chimica Sinica, 2024, 40(10): 2310013-. doi: 10.3866/PKU.WHXB202310013
12. [12]
  Changjun You , Chunchun Wang , Mingjie Cai , Yanping Liu , Baikang Zhu , Shijie Li . 引入内建电场强化BiOBr/C₃N₅ S型异质结中光载流子分离以实现高效催化降解微污染物. Acta Physico-Chimica Sinica, 2024, 40(11): 2407014-. doi: 10.3866/PKU.WHXB202407014
13. [13]
  Qilu DU , Li ZHAO , Peng NIE , Bo XU . Synthesis and characterization of osmium-germyl complexes stabilized by triphenyl ligands. Chinese Journal of Inorganic Chemistry, 2024, 40(6): 1088-1094. doi: 10.11862/CJIC.20240006
14. [14]
  Zhuoming Liang , Ming Chen , Zhiwen Zheng , Kai Chen . Multidimensional Studies on Ketone-Enol Tautomerism of 1,3-Diketones By ¹H NMR. University Chemistry, 2024, 39(7): 361-367. doi: 10.3866/PKU.DXHX202311029
15. [15]
  Xiao SANG , Qi LIU , Jianping LANG . Synthesis, structure, and fluorescence properties of Zn(Ⅱ) coordination polymers containing tetra-alkenylpyridine ligands. Chinese Journal of Inorganic Chemistry, 2024, 40(11): 2124-2132. doi: 10.11862/CJIC.20240158
16. [16]
  Chongjing Liu , Yujian Xia , Pengjun Zhang , Shiqiang Wei , Dengfeng Cao , Beibei Sheng , Yongheng Chu , Shuangming Chen , Li Song , Xiaosong Liu . Understanding Solid-Gas and Solid-Liquid Interfaces through Near Ambient Pressure X-Ray Photoelectron Spectroscopy. Acta Physico-Chimica Sinica, 2025, 41(2): 100013-. doi: 10.3866/PKU.WHXB202309036
17. [17]
  Ruilin Han , Xiaoqi Yan . Comparison of Multiple Function Methods for Fitting Surface Tension and Concentration Curves. University Chemistry, 2024, 39(7): 381-385. doi: 10.3866/PKU.DXHX202311023
18. [18]
  Jinfeng Chu , Yicheng Wang , Ji Qi , Yulin Liu , Yan Li , Lan Jin , Lei He , Yufei Song . Comprehensive Chemical Experiment Design: Convenient Preparation and Characterization of an Oxygen-Bridged Trinuclear Iron(III) Complex. University Chemistry, 2024, 39(7): 299-306. doi: 10.3866/PKU.DXHX202310105
19. [19]
  Aidang Lu , Yunting Liu , Yanjun Jiang . Comprehensive Organic Chemistry Experiment: Synthesis and Characterization of Triazolopyrimidine Compounds. University Chemistry, 2024, 39(8): 241-246. doi: 10.3866/PKU.DXHX202401029
20. [20]
  Zhiwen HU , Weixia DONG , Qifu BAO , Ping LI . Low-temperature synthesis of tetragonal BaTiO₃ for piezocatalysis. Chinese Journal of Inorganic Chemistry, 2024, 40(5): 857-866. doi: 10.11862/CJIC.20230462

Get Citation

PDF

XML

Metrics

PDF Downloads(0)
Abstract views(674)
HTML views(104)

通讯作者: 陈斌, bchen63@163.com

1.
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

Metrics

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