title: A novel protocol for enzymatic digestion based on covalent binding by protein immobilization.
abstract: 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. Graphical abstract Workflow of the IPD method, including protein denaturation, immobilization, digestion, and identification.
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Qi
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China.
authors.forename: Guoquan
authors.initials: G
authors.lastname: Yan
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China.
authors.forename: Chunhui
authors.initials: C
authors.lastname: Deng
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China. xmzhang@fudan.edu.cn.
authors.forename: Xiangmin
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005240:Feasibility Studies; D055767:Immobilized Proteins / Q000737:chemistry*; D066298:In Vitro Techniques; D008099:Liver / Q000737:chemistry; D058185:Magnetite Nanoparticles / Q000737:chemistry; D051379:Mice; D010455:Peptides / Q000032:analysis; D011489:Protein Denaturation; D059748:Proteolysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D055767:Immobilized Proteins; D058185:Magnetite Nanoparticles; D010455:Peptides; D020543:Proteome
keywords: Covalent binding; Denaturation; Functional microspheres; Protein digestion