Method for protein gel internal enzymolysis

Abstract

The method includes following procedures: (1) using ultra pure water to wash up gel containing target protein; dehydrating and drying gel; (2) using treatment fluid of acidized trypsin to soak dryed gel for 0.5-1.5h to absorb unwanted enzyme treatment fluid; the treatment fluid of acidized trypsin is obtained through following steps: using 80-120mM hydrochloric acid to dissolve trypsin, acidizing it for 8-12 minutes, using NH4HCO3 to adjust pH to 8.0-9.0; (3) adding buffer solution containing Ca2+, carrying out enzymolysis at 37 deg.C for 14-18h, removing gel to obtain proteolysis fluid. Reducing difficulty of operation, simplifying experimental procedure, lowering cost, the method raises efficiency of enzymolysis effectively, obtains more information of peptide segment, and higher point value of searching database, and raises percentage of coverage of sequence markedly so as to obtain better qualification result of mass spectrum finally.

Description

technical field [0001] The invention relates to a method for in-gel enzymolysis of proteins suitable for mass spectrometry analysis. Background technique [0002] With the completion of large-scale biological genome sequencing, human beings have entered the proteome era. Currently in proteome research, mass spectrometry (MS) technology is the core protein identification technology (Cánovas F M, Eliane D G, Recorbet G, Jorrin J, Mock H P and Rossignol M. Plant proteome analysis. Proteomics, 2004, 4: 285- 298; Chen S and Harmon A C. Advances in plant proteomics. Proteomics, 2006, 6:5504-5516). According to different ionization methods, mass spectrometry is mainly divided into matrix-assisted laser desorption / ionization time-of-flight (MALDI TOF) and electrospray ionization time-of-flight (ESI) -TOF) two types (Cánovas F M, Eliane D G, Recorbet G, Jorrin J, Mock H P and Rossignol M. Plant proteomeanalysis. Proteomics, 2004, 4: 285-298). Usually, the protein mixture is separa...

AI Technical Summary

Problems solved by technology

However, while obtaining as many peptides as possible, the impurities in the gel are also extracted together, which seriously interferes with the effective ionization process of protein samples during mass spectrometry identification, resulting in peak loss

Therefore, using this method to carry out enzymatic hydrolysis experiments also has shortcomings such as insufficient enzymatic hydrolysis, serious loss of peptide segments, large enzyme consumption, cumbersome operation, etc., and the final database search results are not ideal.

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