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Method of determining position of disulfide bond of polypeptide

A disulfide bond and predetermined time technology, applied in the field of analytical chemistry, can solve the problems of inability to sequence peptides and proteins, and the method needs to be improved, so as to achieve the effects of less difficulty in analysis, less harm to the human body, and simplified procedures

Pending Publication Date: 2020-10-16
NAT INST OF METROLOGY CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Edman degradation cannot directly sequence peptides and proteins containing intrachain disulfide bonds and N-termini blocked
[0004] Therefore, the method of determining the position of the disulfide bond needs to be improved

Method used

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  • Method of determining position of disulfide bond of polypeptide
  • Method of determining position of disulfide bond of polypeptide
  • Method of determining position of disulfide bond of polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] In this embodiment, the confirmation of the disulfide bond in linaclotide is taken as an example to verify the accuracy and simplicity of the method for determining the position of the polypeptide disulfide bond in the embodiment of the present invention. Among them, linaclotide The structure of the peptide is detailed in figure 1 , The specific method is as follows:

[0070] 1. The first liquid chromatography tandem mass spectrometry detection In order to obtain a better signal, the sheath gas flow rate, auxiliary gas flow rate, spray voltage, capillary temperature and NCE energy of the MS MS fragmentation in ESI were optimized. Through the needle pump direct injection optimization experiment, the sheath gas flow rate in ESI is 35arb, the auxiliary gas flow rate is 1arb, the spray voltage is +3.1kv, and the capillary temperature is 310°C. The signal response is better; the fragmented NCE energy of the MS MS is more effective for HCD. 23% is preferred, and for CID, 20% is ...

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Abstract

The invention discloses a method for determining the position of a disulfide bond of polypeptide, which comprises the following steps: carrying out first liquid chromatography-tandem mass spectrometrydetection on a polypeptide sample to obtain a first liquid chromatography-tandem mass spectrometry detection result; mixing the polypeptide sample with a reducing agent and an organic acid solution to obtain a partial reduction product in a first preset time and a complete reduction product in a second preset time; carrying out second liquid chromatography-tandem mass spectrometry detection on the partial reduction product to obtain a second liquid chromatography-tandem mass spectrometry detection result; performing third liquid chromatography-tandem mass spectrometry detection on the complete reduction product to obtain a third liquid chromatography-tandem mass spectrometry detection result; and determining the position of the disulfide bond of the polypeptide based on the first liquid chromatography tandem mass spectrometry detection result, the second liquid chromatography tandem mass spectrometry detection result and the third liquid chromatography tandem mass spectrometry detection result. The method does not need alkylation, and the positioning method is simple, rapid, green and easy to operate.

Description

Technical field [0001] The present invention relates to the field of analytical chemistry, in particular, to a method for determining the position of a polypeptide disulfide bond. Background technique [0002] Disulfide bond is a very important post-translational modification formed by the covalent cross-linking of two cysteine ​​side chain sulfhydryl groups (-SH) within or between polypeptide chains. In the field of biomedicine, more and more proteins or peptides are used as biological agents or vaccines to treat various diseases. These biological agents include hormones, growth factors, and monoclonal antibodies. Most of these biological molecules contain disulfide bonds. Drugs containing disulfide bonds are prone to exchange and rearrangement of disulfide bonds during synthesis. These side reactions can lead to the formation of non-native disulfide bonds and protein aggregation, which may induce immunogenicity and inactivate the drug. Therefore, the location and analysis of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/72G01N30/06G01N30/88
CPCG01N30/02G01N30/72G01N30/06G01N30/88G01N2030/067G01N2030/8813
Inventor 吴佩泽李明李红梅
Owner NAT INST OF METROLOGY CHINA