Analysis method for determining modification sites of multi-site PEG modified protein
A protein modification, multi-site technology, applied in the field of protein modification site analysis, can solve the problems of difficult site judgment and heavy workload.
Active Publication Date: 2019-08-13
CHONGQING PEG BIO BIOTECH CO LTD
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Problems solved by technology
[0013] In order to improve the accuracy of the judgment of the modified site and alleviate the problem of difficult site judgment and heavy workload due to the inhomogeneity of polyethylene glycol, the present invention provides a method for determining the modification site of a multi-site PEG-modified protein analysis method
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[0036] The following are non-limiting examples of the present invention, which will help those of ordinary skill in the art to further understand the present invention.
[0037] This study mainly refers to the principle of multi-enzyme digestion to determine the modification site analysis described in the content of the invention, and adopts the method of more than two enzyme digestions, that is, the first enzyme digestion to determine the modified peptide, recover and concentrate the modified peptide, and After the peptide was digested twice, LC-MS analysis was performed to determine the modification site. The specific experimental method is established as follows:
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A modified site analysis technology for multi-site modification disclosed by the invention realizes the localization of PEGylated protein modification sites for four or more modification sites, and avoids direct measurement of PEG-modified peptides impossible to measure molecular weight, while the peptides not containing modified sites are performed with accurate measurement of molecular weight after the specificity of the PEG-modified peptides is recovered and enzyme-cut again, and accurate PEG-modified sites are obtained by comparison with theoretical peptides. The method realizes the determination of more modified sites of protein of the modified sites with high average modification degree, has specificity to the PEG-modified peptides, simplifies the detection steps, reduces the workload, and is convenient to operate, easy to implement and high in repetition rate.
Description
technical field [0001] The invention belongs to a method for analyzing the structure of a protein after modification, in particular to a method for analyzing a protein modification site. Background technique [0002] Protein drugs mainly include proteins with special functions such as peptides, enzymes, cytokines, hormones, and antibodies. With the development of biotechnology, people can obtain the required drugs through fermentation engineering, genetic engineering, protein engineering, and enzyme engineering. protein. However, a large number of clinical studies have found that drugs without any modification have a series of defects in vivo, such as short half-life, poor stability, easy to be degraded by enzymes in vivo, and strong immunogenicity. For this reason, people try to modify protein drugs in various ways to adapt to clinical applications. [0003] Among them, polyethylene glycol (PEG) is a polymer of repeated chemical structure ethylene glycol. There are a larg...
Claims
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Login to View More IPC IPC(8): G01N30/88C12Q1/37
CPCC12Q1/37G01N30/88G01N2030/8831
Inventor 胡春兰陈清付志成栾秋洋范开
Owner CHONGQING PEG BIO BIOTECH CO LTD
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