Protein separating and enriching method
A technology for separation and enrichment, protein, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc.
Inactive Publication Date: 2014-03-05
刘军
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Problems solved by technology
So far, there is still no good method that
Method used
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[0009] Firstly, let the sample go through the first lectin affinity chromatography, then perform enzymatic hydrolysis, then subject the sample to the second lectin affinity chromatography, and finally use HPLC to separate and enter the mass spectrometer for sequencing. The lectins used include concanavalin A, malt agglutinin, bean agglutinin, etc.; the processed samples are analyzed for glycopeptides by capillary liquid chromatography-ESI-MASS or capillary liquid chromatography-MADLI-TOF-MASS and database searches.
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The invention discloses a protein separating and enriching method. The method is characterized by comprising the following steps: subjecting a sample to a primary lectin affinity chromatography, then carrying out an enzymatic hydrolysis on the sample, then subjecting the sample to a secondary lectin affinity chromatography, finally separating the sample by utilizing HPLC, and sequencing with a mass spectrum instrument. The agglutinin used in the method comprises concanavalin A, wheat germ agglutinin, PHA phaseolus vulgaris agglutinin, etc. The glycopeptides in the processed sample is analyzed and retrieved in a database by a capillary liquid chromatography-ESI-MASS or a capillary liquid chromatography-MADLI-TOF-MASS. The method has the advantages that various saccharide species can be collected in one time, the labeled polypeptide can be enriched through an affinity method, and a quantitative and comparative analysis is advantageously to be achieved through adopting an isotope labeled reagent.
Description
technical field [0001] The invention relates to a biochemical experimental method, specifically, a method for separating and enriching proteins. Background technique [0002] At present, protein drugs produced by genetic engineering have attracted more and more attention from researchers due to their wide application in the treatment of tumors and immune diseases. For the protein drug itself, the realization of its important function depends not only on the sequence and spatial structure of its protein, but also on factors such as protein post-translational modification, among which glycosylation modification is one of the most important modification methods in post-translational modification . The detection and analysis of protein glycosylation is also an important index to measure protein drugs. Glycosylation analysis of protein mainly includes the following aspects: whether glycosylation occurs; where glycosylation occurs; what type of glycosylation is; level structure...
Claims
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Inventor 刘军
Owner 刘军
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