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
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0052] In order to fully illustrate the technical solution of the present invention, an example is used to locate the connection mode of disulfide bonds in a polypeptide (molecular weight 3140) composed of 30 amino acids (6 cysteines among which form disulfide bonds with each other), and its primary structure Such as figure 1 shown.
[0053] The first step, partial reduction of the disulfide bond of the polypeptide sample to be tested
[0054]Add 100 μL of 0.5M DTT solution (pH 6.5) to 2 mg of sample and incubate for 30 min (60°C), add 10 μL of TCEP (0.2M) solution, react at 60°C for 20 min, add 50 μL of 0.2% TFA aqueous solution to terminate the reaction. Separation was carried out on 515HPLC of Waters Company in the United States, the separation column was Kromasil 250*4.6mm C18 column, and the separation gradient was as follows:
[0055] time (min) Flow rate (mL / min) A% (0.1% TFA / H 2 O) B% (0.1% TFA / H 2 O) 0 1 80 20 40 1 60 40 ...
Embodiment 2
[0063] The second step is to alkylate the uncycled cysteine sulfhydryl groups of peak A and peak B
[0064] React the freeze-dried sample of peak A with 50 μl of 1.0 M iodoacetamide solution at 60°C for 10 minutes, add 100 μl of 0.2% TFA aqueous solution to terminate the reaction, and separate on 515HPLC of Waters Company in the United States. The gradient is as before, and the chromatogram is as follows Figure 7 shown.
[0065] Collect 10.14min elution peak, adopt MALDI-TOF mass spectrum identification, its molecular weight is 3376 (see Figure 8 ).
[0066] The results of mass spectrometry showed that the molecular weight of the chromatographic peak with a retention time of 10.14min was 232Da more than that of peak A before being alkylated, indicating that four semi-light amino acid residues were modified by alkylation.
[0067] React the lyophilized sample of peak B with 50 μl of 1.0 M iodoacetamide solution at 60°C for 10 minutes, add 100 μl of 0.2% TFA aqueous s...
Embodiment 3
[0071] Step 3: Break all disulfide bonds in the polypeptide chain (all reduction)
[0072] The partially reduced product of the alkylation was reacted with 1M dithiothreitol (DTT) at 50°C for 20 minutes to break all the disulfide bonds in the polypeptide chain, and samples were taken for mass spectrometry identification after the reaction.
[0073] Mass spectrometry results show that the molecular weight of the alkylated A peak eluate is 3378 after the reaction, which is 2 Da more than before the reaction, indicating that a pair of disulfide bonds are broken (see Figure 11 ).
[0074] Mass spectrometry results show that the molecular weight of the alkylated peak B eluate after reaction is 3262, which is 4Da more than before the reaction, indicating that there are two pairs of disulfide bonds broken (see Figure 12 ).
PUM
![No PUM](https://static-eureka.patsnap.com/ssr/23.2.0/_nuxt/noPUMSmall.5c5f49c7.png)
Abstract
Description
Claims
Application Information
![application no application](https://static-eureka.patsnap.com/ssr/23.2.0/_nuxt/application.06fe782c.png)
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com