Positioning method of disulfide bonds in polypeptide

A positioning method and disulfide bond technology, applied in measuring devices, material analysis through electromagnetic means, instruments, etc., can solve problems such as laborious, unstable analysis results, time-consuming, etc., and achieve simple operation, accurate results, and integrity maintenance Effect
CN102721734AActive Publication Date: 2012-10-10HYBIO PHARMA
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
HYBIO PHARMA
Publication Date
2012-10-10

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Abstract

The invention relates to a determination method of a polypeptide structure, in particular to a method for positioning a disulfide bond connecting structure in polypeptide. The method comprises the steps of: 1) incubating polypeptide to be determined and dithiothreitol at high temperature, adding TCEP (Trichloroethyl Phosphate) to react, and separating to obtain partially-reduced product; 2) performing reaction of the partially-reduced product with iodoacetamide at high temperature so as to alkylate sulfydryl in a disconnection part of the disulfide bond; and separating to obtain partially alkylated reduzate; 3) performing reaction of the partially alkylated reduzate with dithiothreitol so as to disconnect all disulfide bonds in the partially alkylated reduzate; and 4) performing detective analysis by matrix-assisted laser desorption ionization-time-of-flight mass spectrum and electrospray-quadrupole rod-time-of-flight mass spectrum to position the disulfide bond connecting structure. According to the positioning method of the disulfide bonds in polypeptide provided by the invention, a partial reduction method combined with MS / MS is adopted to position the disulfide bonds so that the method is simple and convenient to operate, high in efficiency and accurate in result.
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Description

technical field

[0001] The invention relates to a method for determining the structure of a polypeptide, in particular to a method for locating disulfide bonds in the polypeptide. Background technique

[0002] When a polypeptide chain contains multiple cysteines and forms multiple pairs of disulfide bonds, its side chain sulfhydryl groups may have multiple connection methods. How to determine the pairing method of sulfhydryl groups is a very important technology in the field of polypeptide research. In the published literature and patents, there are mainly the following methods for the positioning of disulfide bonds in polypeptides: 1) Enzyme digestion, 2) Diagonal electrophoresis, 3) Partial reduction combined with Edman degradation, Edman degradation sequencing, This method is time-consuming, labor-intensive, and the analytical results are unstable. 4) X-ray crystallography, 5) two-dimensional NMR, etc., but the method of partial reduction combined with MS / MS to locate di...

Claims

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