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Rapid protein identification using monolithic enzymatic microreactor and LC-ESI-MS/MS

Authors

  • Jicheng Duan,

    1. National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, P. R. China
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  • Zhen Liang,

    1. National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, P. R. China
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  • Chun Yang,

    1. National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, P. R. China
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  • Jie Zhang,

    1. National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, P. R. China
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  • Lihua Zhang,

    1. National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, P. R. China
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  • Weibing Zhang,

    1. National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, P. R. China
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  • Yukui Zhang Professor

    Corresponding author
    1. National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, P. R. China
    • National Chromatographic Research & Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, P. R. China Fax: +86-411-84379540
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Abstract

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. In addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.

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