A novel protocol for enzymatic digestion based on covalent binding by protein immobilization

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• 作者：Qian Qi ; Guoquan Yan ; Chunhui Deng…
• 关键词：Protein digestion ; Denaturation ; Functional microspheres ; Covalent binding
• 刊名：Analytical and Bioanalytical Chemistry
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：408
• 期：29
• 页码：8437-8445
• 全文大小：2,169 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Analytical Chemistry
  Food Science
  Inorganic Chemistry
  Physical Chemistry
  Monitoring, Environmental Analysis and Environmental Ecotoxicology
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1618-2650
• 卷排序：408

文摘

The process of protein digestion is a critical step for successful protein identification in proteomic analysis. Many efforts have been dedicated to enhancing the digestion efficiency for sufficient digestion. Among these approaches, protein complete denaturation with denaturants is a common process for better digestion. However, the removal of denaturants was tedious or would cause protein loss and other problems. In this work, a feasible digestion approach, immobilized protein digestion (IPD), based on covalent binding has been developed. Proteins can be completely denatured and immobilized on the surface of functional materials by covalent binding to form a monolayer. Subsequently, varieties of denaturants or contaminants would be removed thoroughly by washing. To achieve fast immobilization and high digestion efficiency, different functional materials and denaturants were selected. Compared with traditional in-solution digestion, the method achieved a prominent increase in identified peptides numbers and sequence coverage of proteins. Data analysis also showed that covalent binding could evidently decrease enzymatic missed cleavage for various protein sequences. Furthermore, possible peptide losses due to covalent binding were also investigated. Also, it has been proved to be efficient for complex biological sample digestion.