Investigation of bi-enzymatic reactor based on hybrid monolith with nanoparticles embedded and its proteolytic characteristics.
The bottom-up strategy of proteomic profiling study based on mass spectrometer (MS) has drawn high attention. However, conventional solution-based digestion could not satisfy the demands of highly efficient and complete high throughput proteolysis of complex samples. We proposed a novel bi-enzymatic reactor by immobilizing two different enzymes (trypsin/chymotrypsin) onto a mixed support of hybrid organic-inorganic monolith with SBA-15 nanoparticles embedded. Typsin and chymotrypsin were crossly immobilized onto the mixed support by covalent bonding onto the monolith with glutaraldehyde as bridge reagent and chelation via copper ion onto the nanoparticles, respectively. Compared with single enzymatic reactors, the bi-enzymatic reactor improved the overall functional analysis of membrane proteins of rat liver by doubling the number of identified peptides (from 1184/1010 with trypsin/chymotrypsin enzymatic reactors to 2891 with bi-enzymatic reactor), which led to more proteins identified with deep coverage (from 452/336 to 620); the efficiency of the bi-enzymatic reactor is also better than that of solution-based tandem digestion, greatly shorting the digestion time from 24h to 50s. Moreover, more transmembrane proteins were identified by bi-enzymatic reactor (106) compared with solution-based tandem digestion (95) with the same two enzymes and enzymatic reactors with single enzyme immobilized (75 with trypsin and 66 with chymotrypsin). The proteolytic characteristics of the bi-enzymatic reactors were evaluated by applying them to digestion of rat liver proteins. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weight.
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, P.R. China.
Lulu
L
Shangguan
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, P.R. China. Electronic address: zhanglingyi@ecust.edu.cn.
Lingyi
L
Zhang
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, P.R. China.
Zhichao
Z
Xiong
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, P.R. China.
Jun
J
Ren
Shanghai Key Laboratory of Crime Scene Evidence, Shanghai Research Institute of Criminal Science and Technology, Shanghai Public Security Bureau, Shanghai, P.R. China.
Runsheng
R
Zhang
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, P.R. China.
Fangyuan
F
Gao
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, P.R. China.
Weibing
W
Zhang
D000818:Animals; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008099:Liver / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry*; D059748:Proteolysis; D051381:Rats; D012822:Silicon Dioxide / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D004800:Enzymes, Immobilized; D008565:Membrane Proteins; C509969:SBA-15; D012822:Silicon Dioxide; D002918:Chymotrypsin; D014357:Trypsin
Bi-enzymatic reactor; Hybrid organic–inorganic monolith; Membrane protein; Nanoparticle; Proteolysis