title: Preparation and application of immobilized enzymatic reactors for consecutive digestion with two enzymes.
abstract: The bottom up strategy has drawn much attention due to the high accuracy, reliability, and reproducibility in protein identification in which proteins are digested into peptides. However, conventional solution-based digestion and enzymatic reactor with one protease immobilized cannot satisfy high throughput proteolysis of complex samples. Application of consecutive hydrolysis by enzymatic reactor can be a new strategy for high throughput proteolysis of complex samples by adjusting immobilization amount of the enzymes, enzyme ratio, as well as hydrolysis order of two enzymes. In this work, we propose immobilized enzymatic reactor for consecutive digestion with two enzymes by combining two enzyme reactors with trypsin and chymotrypsin immobilized, respectively. Each reactor was prepared individually by immobilizing only one protease (trypsin or chymotrypsin) to hybrid monolith with SBA-15 particles embedded. Proteolysis conditions including hydrolysis order and trypsin to chymotrypsin ratio etc. were studied using standard proteins. Best digestion performance was obtained when the proteins were digested by trypsin first with trypsin to chymotrypsin ratio of 1:1. When applying them to digestion of rat liver proteins, total 1651 proteins and 11011 peptides were identified by combining four enzymolysis strategies with two enzymes including proteolytic digestion in two consecutive enzymatic reactors, synergy enzymolysis with two enzymes in one immobilized enzymatic reactor and consecutive hydrolysis with two enzymes in-solution digestion respectively, in which consecutive enzymolysis in enzymatic reactors gave the best results with 1091 proteins and 5071 peptides identified. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weights, and will play an important role in high efficient and high throughput proteomics research.
authors.affiliation: Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Bingbing
authors.initials: B
authors.lastname: Wang
authors.affiliation: Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Lulu
authors.initials: L
authors.lastname: Shangguan
authors.affiliation: Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Shulei
authors.initials: S
authors.lastname: Wang
authors.affiliation: Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, PR China; Center of Analysis and Testing, Nanchang University, Jiangxi 330047, PR China. Electronic address: zhanglingyi@ecust.edu.cn.
authors.forename: Lingyi
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Weibing
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Center of Analysis and Testing, Nanchang University, Jiangxi 330047, PR China.
authors.forename: Fan
authors.initials: F
authors.lastname: Liu
mesh_terms: D000818:Animals; D019149:Bioreactors; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006868:Hydrolysis; D008099:Liver / Q000378:metabolism; D008970:Molecular Weight; D011506:Proteins / Q000378:metabolism*; D059748:Proteolysis; D040901:Proteomics; D051381:Rats; D012015:Reference Standards; D015203:Reproducibility of Results; D012822:Silicon Dioxide / Q000737:chemistry; D012996:Solutions; D014357:Trypsin / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D011506:Proteins; C509969:SBA-15; D012996:Solutions; D012822:Silicon Dioxide; D002918:Chymotrypsin; D014357:Trypsin
keywords: Complex protein samples; Consecutive proteolytic digestion; Enzymatic characteristics; Immobilized enzymatic reactor (IMER)