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Efficient Proteolysis of Glycoprotein Using a Hydrophilic Immobilized Enzyme Reactor Coupled with MALDI-QIT-TOF-MS Detection and μHPLC Analysis

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Abstract

A hydrophilic immobilized enzyme reactor (IMER) containing trypsin was prepared and applied in the proteolysis of glycoproteins. Glycoproteins including horseradish peroxidase, asialofetuin, and fetuin were used to evaluate the performance of the hydrophilic IMER for the glycoprotein digestion. The digested products were detected by matrix-assisted laser desorption/ionization quadruple ion trap time-of-flight mass spectrometry and micro-high-performance liquid chromatography. The hydrophilic IMER showed higher enzymatic digestion efficiency compared with conventional in-solution digestion. The digestion time could be reduced from 16 h to several minutes. Furthermore, using microwaves as a heat source, the reproducibility of the hydrophilic IMER was evaluated and this IMER could be recycled for at least ten times without obvious loss of enzyme activity. The hydrophilic IMER provides a promising tool for high-throughput glycoproteome analysis.

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Acknowledgments

This work was supported by the Project 2012YQ12004409, the 863 Project: 2012AA020202, 973 Project: 2012CB916604 and the National Natural Science Foundation Program, Project 21175026.

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Authors and Affiliations

  1. Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, People’s Republic of China

    Chaofeng Wang, Mingxia Gao, Peng Zhang & Xiangmin Zhang

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  1. Chaofeng Wang
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  2. Mingxia Gao
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  3. Peng Zhang
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  4. Xiangmin Zhang
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Correspondence to Xiangmin Zhang.

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Wang, C., Gao, M., Zhang, P. et al. Efficient Proteolysis of Glycoprotein Using a Hydrophilic Immobilized Enzyme Reactor Coupled with MALDI-QIT-TOF-MS Detection and μHPLC Analysis. Chromatographia 77, 413–418 (2014). https://doi.org/10.1007/s10337-013-2622-4

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  • DOI: https://doi.org/10.1007/s10337-013-2622-4

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