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

Active Publication Date: 2012-10-10
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

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  • Positioning method of disulfide bonds in polypeptide
  • Positioning method of disulfide bonds in polypeptide
  • Positioning method of disulfide bonds in polypeptide

Examples

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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 ).

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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.

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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Application Information

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IPC IPC(8): G01N27/62
Inventor 康旭宓鹏程马亚平袁建成
Owner HYBIO PHARMA
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