| As important part in " bottom-up" strategy of proteomics, immobilized enzyme reactor has great significance to develop faster and more efficient methods of protein analysis. A novel immobilized enzyme reactor was prepared by combining nanoparticles with organic-inorganic hybrid monolith. It can achieve high digestion efficiency owing to making use of the advantages of nanoparcticles and organic-inorganic hybrid monolith including high specific surface area, easy controllable size, simple operation, fast mass transfer and easy modification.Nanoparticles modified with amnio was embedded in organic-inorganic hybrid monolith using tetraethoxysilane (TEOS) and3-aminopropyltriethoxysilane (APTES) as monomer. A trypsin reactor based on hybrid monolith with nanoparticles incorporated was obtained by covalent trypsin with glutaraldehyde as bridging reagent. At the best conditions, this trypsin reactor obtained sequence coverage of50%,93%,71%for bovine serum albumin, myoglobin, and Cytochrome C and quickly digested complex sample of milk and rat liver protein. The trypsin reactor shows great potential for high througput analysis of complex proteins.Combination of different enzymes will increase the identification confidence of protein in virtue of their different specific cutting sites. Thus, a bi-enzymes reactor based on monlith with nanoparticles incorporated was prepared after embedding nanoparticles modfied with carboxy into organic-inorganic hybrid monolith. The monolith and nanoparticles were covalently modified with trypsin using glutaraldehyde as bridging reagent and chelated with chymotrypsin through copper ion, respectively. At the best conditions,536kinds of protein were identified by RPLC-MS after digesting by this bi-enzymes reactor in hydrophobic membrane protein extracted from rat liver. Compared with single-enzyme reactor, the bi-enzymes reactor presentes better performance and provides a new possible way for proteomics research. |
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