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Non-reducing peptide mapping method of protein

An analytical method and protein technology, applied in the field of biological proteins, can solve the problems of incomplete enzymatic hydrolysis, few peaks, and poor separation of chromatographic peaks, and achieve good separation, more peaks, and stable chromatographic baseline. Effect

Active Publication Date: 2019-12-27
CHENGDU KANGHONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve a series of defects such as incomplete enzymatic hydrolysis, poor separation of chromatographic peaks, and a small number of peaks in the prior art protein peptide map analysis, the present invention provides the following peptide map analysis method on the one hand, the steps include: :

Method used

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  • Non-reducing peptide mapping method of protein

Examples

Experimental program
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Effect test

Embodiment 1

[0121] The protein sample treatment is as follows: take 0.2mg of the protein sample containing sequence 1, use 400μl of ultrapure water each time, 14000g, and change the liquid for 5min ultrafiltration three times, collect the protein solution after ultrafiltration with a pipette, if the solution volume is less than 50μl, use ultrapure water Make up with water, then add 50μl 0.2w / v% RapiGest SF and mix well, place the mixture in a boiling water bath for 10min, then take it out and cool to room temperature; add 20μl of 0.5M ammonium bicarbonate solution and 100μl 0.1mg / ml Promega Sequencing-grade trypsin, after mixing, take it out in a 37°C water bath for 16-18h, and occasionally take it out and shake gently; after the 37°C water bath, add 22 μl of 50% acetic acid solution, centrifuge at 10,000×g for 5 min, and take the supernatant for testing.

[0122] Negative samples are processed as follows: take 50 μl ultrapure water, 50 μl 0.2% RapiGest SF, mix well, place in a boiling wat...

Embodiment 2

[0137] Except that the mobile phase B is: 0.085% trifluoroacetic acid in acetonitrile solution, wherein the acetonitrile solution contains 10% water, other conditions are the same as in Example 1.

[0138] Test results:

[0139] Test results such as Figure 6 and Table 6, from Figure 6 As can be seen from Table 6, when the water content in the mobile phase B is 10%, the number of peaks in the elution chromatogram is more (75 chromatographic peaks), the distribution of the chromatographic peaks of each peptide is relatively uniform, and the baseline is relatively stable.

[0140] Table 6 Protein peptide map detection results

[0141]

[0142]

Embodiment 3

[0144] Except that the mobile phase B is: 0.085% trifluoroacetic acid in acetonitrile solution, wherein the acetonitrile solution contains 30% water, other conditions are the same as in Example 1.

[0145] Test results:

[0146] The protein sample test results are as follows: Figure 7 and Table 7, from Figure 7 As can be seen from Table 7, the number of peaks in the elution chromatogram is large (56 chromatographic peaks), the distribution of the chromatographic peaks of each peptide is relatively uniform, and can be separated better, and the baseline is relatively stable.

[0147] Table 7 Protein peptide map detection results

[0148]

[0149]

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Abstract

The invention provides a non-reducing peptide mapping method of a protein. The method comprises the following steps: denaturing the protein by using a denaturing agent, carrying out enzymolysis, terminating the reaction with an acid, and performing detection by reversed phase high-performance liquid chromatography. The non-reducing peptide mapping method makes enzymolysis of the protein sufficient, realizes uniform distribution, large number and good resolution of chromatographic peaks in a map obtained by the reversed phase high-performance liquid chromatography detection, allows the chromatogram to have a stable baseline and truly reflect the situation of each peptide fragment obtained after the enzymolysis of the protein, and is of great significance to controlling the quality of the protein.

Description

technical field [0001] The invention relates to the field of biological proteins, in particular to a non-reducing peptide map analysis method for proteins. Background technique [0002] Peptide Mapping is based on the molecular weight and amino acid composition characteristics of proteins and polypeptides, using specific proteolytic enzymes (generally endopeptidases) to act on specific peptide chain sites Cleavage the polypeptide into small fragments, and form a characteristic fingerprint through certain separation and detection means. Peptide map analysis is of great significance to the study of peptide structure and identification of characteristics. Utilizing the property that trypsin can specifically act on the peptide chains at the carboxy-terminals of Arg and Lys, the characteristic trypsin spectrum of the protein was detected by RP-HPLC using a C18 column as an identification analysis. Peptide maps can identify single amino acid changes due to complementary DNA read...

Claims

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Application Information

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IPC IPC(8): G01N30/89
CPCG01N30/89
Inventor 柯潇唐懿挺罗祖秀
Owner CHENGDU KANGHONG BIOTECH
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