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

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• 作者：Qian ZHANG ; Fei ZHENG ; Wei-Jie QIN ; ^{qianxh1@163.com" class="auth_mail" title="E-mail the corresponding author} ; Xiao-Hong QIAN ; ^{aunp_dna@126.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Immobilized enzyme ; Thermo-sensitive polymer ; Mass spectrometry ; Proteomics ; Fast digestion
• 刊名：Chinese Journal of Analytical Chemistry
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：44
• 期：11
• 页码：1692-1697
• 全文大小：717 K

文摘

By massively analyzing proteins extracted from cells, tissues and organisms using mass spectrometry, proteomics is capable of providing information about the 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 change. The thermo-sensitive immobilized trypsin had the feature of “homogeneous digestion at high temperature and heterogeneous separation at low temperature” and the advantages of significantly shortened digestion time and recover & reuse of the enzyme. When BSA was digested 1 min by immobilized enzyme, the amino acid sequence coverage was up to 94%, higher than 74% amino acid sequence coverage in traditional enzyme solution. Finally, the immobilized-trypsin was successfully applied in fast and highly efficient digestion of complex proteome extracted from HeLa cell. The efficiency of immobilized enzyme 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.