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SDS-PAGE-free protocol for comprehensive identification of cytochrome P450 enzymes and uridine diphosphoglucuronosyl transferases in human liver microsomes

Authors

  • Liangliang Sun,

    1. Laboratory of High Efficient Separation and High Sensitive Characterization of Biomolecules, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China
    Current affiliation:
    1. Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, USA
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  • Yanyan Zhang,

    Corresponding author
    1. Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China
    • Laboratory of High Efficient Separation and High Sensitive Characterization of Biomolecules, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China
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  • Dingyin Tao,

    1. Laboratory of High Efficient Separation and High Sensitive Characterization of Biomolecules, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China
    Current affiliation:
    1. Johns Hopkins Bloomberg School of Public Health, Baltimore, USA
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  • Guijie Zhu,

    1. Laboratory of High Efficient Separation and High Sensitive Characterization of Biomolecules, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China
    Current affiliation:
    1. Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, USA
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  • Qun Zhao,

    1. Laboratory of High Efficient Separation and High Sensitive Characterization of Biomolecules, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China
    2. Graduate School of Chinese Academy of Sciences, Beijing, China
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  • Qi Wu,

    1. Laboratory of High Efficient Separation and High Sensitive Characterization of Biomolecules, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China
    2. Graduate School of Chinese Academy of Sciences, Beijing, China
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  • Zhen Liang,

    1. Laboratory of High Efficient Separation and High Sensitive Characterization of Biomolecules, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China
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  • Ling Yang,

    1. Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China
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  • Lihua Zhang,

    Corresponding author
    • Laboratory of High Efficient Separation and High Sensitive Characterization of Biomolecules, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China
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  • Yukui Zhang

    Corresponding author
    • Laboratory of High Efficient Separation and High Sensitive Characterization of Biomolecules, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China
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Correspondence: Dr. Lihua Zhang, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, China

E-mail: lihuazhang@dicp.ac.cn

Fax: +86-411-84379560

Abstract

Comprehensive identification of cytochrome P450 enzymes (CYPs) and uridine diphosphoglucuronosyl transferases (UGTs) in human liver microsomes (HLMs) was performed with an SDS-PAGE-free protocol. HLMs were solubilized with 5% v/v ionic liquid, 1-butyl-3-methyl imidazolium tetrafluoroborate, followed by tryptic digestion, and 2D-SCX-RPLC-ESI-MS/MS (LTQ XL) analysis in triplicate. In total, 27 CYPs and 12 UGTs were confidently identified with average sequence coverage as 30.99 and 25.07%, average peptide number as 14 and 13, and average unique peptide number as 7 and 4, respectively. The highly similar isoforms of CYP3A, CYP2C, and CYP4F subfamilies could be unambiguously differentiated from each other, despite the fact that the sequence similarity of CYP2C9 and CYP2C19 is 91%. In addition, protein spectral count was used to approximately evaluate the relative abundance of identified CYPs and UGTs, and the results agreed with previous immunochemistry reports.

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