Protease substrate screening method based on solid-loaded mixed protein as screening database

Abstract

The invention discloses a protease substrate screening method based on solid-loaded mixed protein as a screening database. Specifically, mixed protein is bonded onto a solid phase carrier by chemical action to use as the screening database, the protein database is incubated with protease, peptide fragments which are produced by enzyme digestion of substrate protein of the protease in the protein database enter into a solution, non substrate protein cannot be enzyme digested, so that the non substrate protein is completely retained in the solid phase carrier, after solid-liquid separation, qualitative and quantitative identification of the substrate fragments in the solution is performed by bio mass spectrometry so as to obtain the specific protease substrate protein information. The protease substrate screening method is a method using highly specific effects of the protease and a substrate thereof and simplicity of the solid-liquid separation to screen the substrate protein from the mixed protein database.

Description

technical field [0001] The invention is a protease substrate screening method based on immobilized mixed protein as a screening library. Proteases cleave their substrates with high specificity and selectivity. The substrate protein in the protein library immobilized on the solid phase carrier can be digested by protease, and the generated peptides enter the solution, while the non-substrate protein cannot be digested by the enzyme and still remains on the solid phase carrier, through the solid-liquid separation method That is, the enzyme-cleaved peptide of the substrate is selectively separated from the non-substrate protein, and the qualitative and quantitative analysis of the substrate protein is carried out in combination with biological mass spectrometry, so as to obtain the substrate protein of the specific protease. Background technique [0002] As an important regulatory enzyme in life, protease is involved in signal transduction pathways such as cell proliferation, ...

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Problems solved by technology

However, the end-group-labeled enzyme-cleaved peptides produced by this method are interfered by a large number of background peptides, which requires selective enrichment materials and labeling quantification methods, as well as multi-step operations such as sample de

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