Citation: ZHANG Qian, ZHENG Fei, QIN Wei-Jie, QIAN Xiao-Hong. Preparation and Application of Novel Thermo-sensitive Matrix-based Immobilized Enzyme for Fast and Highly Efficient Proteome Study[J]. Chinese Journal of Analytical Chemistry, ;2016, 44(11): 1692-1697. doi: 10.11895/j.issn.0253-3820.160176 shu

Preparation and Application of Novel Thermo-sensitive Matrix-based Immobilized Enzyme for Fast and Highly Efficient Proteome Study

Beijing Institute of Radiation Medicine, National Center for Protein Sciences(Beijing), State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing 102206, China

Received Date: 10 March 2016
Revised Date: 11 July 2016

Fund Project: This work was supported by the National Key Program for Basic Research of China (Nos. 2013CB911204, 2016YFA0501403), the National Key Scientific Instrument Development Program of China (No. 2011YQ09000504), the National High-Tech Research and Development Program of China (No.2014AA020906), and the National Natural Science Foundation of China (Nos. 21275005, 21235001, 21405175 and 21675172)

By massively analyzing proteins extracted from cells, tissues and organisms using mass spectrometry, proteomics is capable of providing information about change in proteins expression, modification, composition and quantification. However, most immobilized enzymes used in mass spectrometry based "shotgun" proteomic strategy are prepared using solid materials as the immobilization matrix and digest the substrate proteins in heterogeneous system. The inherent mass transfer resistance in the solid-liquid interface and steric hindrance of the solid matrix limits the digestion efficiency and sample processing throughput. Here, we prepared a novel immobilized enzyme using soluble thermo-sensitive polymer as the matrix material by exploiting the thermo-responsive ability of the polymer to environmental temperature changes. The thermo-sensitive immobilized trypsin had the feature of "homogeneous digestion at high temperature and heterogeneous separation at low temperature" and the advantage of significantly shortened digestion time and recover and reuse of the enzyme. An amino acid sequence covering up to 94% in 1-min digestion was obtained, which was higher than that of 74% obtained by in-solution digestion for 12 h. Finally, the immobilized trypsin was successfully applied to fast and highly efficient digestion of complex proteome extracted from HeLa cell. The efficiency of immobilized digestion in 1 min was similar to that of solution digestion in 12 h, which sufficiently demonstrated the application potential of this thermo-sensitive immobilized trypsin in proteomics research.
- Immobilized enzyme,
- Thermo-sensitive polymer,
- Mass spectrometry,
- Proteomics,
- Fast digestion
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Preparation and Application of Novel Thermo-sensitive Matrix-based Immobilized Enzyme for Fast and Highly Efficient Proteome Study

南开大学师唯/华北电力大学（保定）刘景维：二维配位聚合物中有序的亲锂冠醚位点用于无枝晶锂沉积

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通讯作者: 陈斌, bchen63@163.com