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Protein C-terminal peptide enrichment method based on Edman degradation

A protein and enrichment technology, applied in the preparation of test samples, etc., can solve the problems of low reaction efficiency and low recovery rate of terminal peptide enrichment, achieve high enrichment selectivity, and improve the effect of enrichment selectivity

Inactive Publication Date: 2017-06-16
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of this method is high selectivity, but the disadvantage is that the reaction conditions need to be strictly controlled to avoid enzyme-catalyzed hydrolysis reactions, resulting in enzyme-linked reactions that cannot be carried out under optimal conditions, and the reaction efficiency is low, so the recovery rate of terminal peptide enrichment is low.

Method used

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  • Protein C-terminal peptide enrichment method based on Edman degradation

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Effect test

Embodiment 1

[0018] Such as figure 1 As indicated, protein C-terminal peptide enrichment was carried out as follows:

[0019] 1) Enzymatic hydrolysis of protein: The two extracted plasma proteins were dissolved in 50mM NaHCO3 (pH7.5), then heat denatured at 90°C for 20min, cooled to room temperature and then reduced (10mM DTT, 56°C, 2h) to alkane Kylation (25mM IAA, protected from light at room temperature, 40min), followed by adding Lys-C at an enzyme / protein ratio of 1:25 for enzymatic hydrolysis and reacting in a 37°C water bath for 16h.

[0020] 2) Edman degradation of protein enzymatic peptides: under alkaline conditions (pH = 8.0), 45 ° C React with the protein enzymatic peptide, the reaction time is 30 minutes. After the reaction mixed solution was lyophilized, 10 microliters to 1 milliliter of anhydrous trifluoroacetic acid was added and reacted at 45° C. for 10 minutes to cleave the N-terminal amino acid of the peptide from the peptide.

[0021] 3) Enrichment of protein C-term...

Embodiment 2

[0023] Such as figure 1 As indicated, protein C-terminal peptide enrichment was carried out as follows:

[0024] 1) Enzymatic hydrolysis of protein: The two extracted plasma proteins were dissolved in 50mM NaHCO3 (pH7.5), then heat denatured at 90°C for 20min, cooled to room temperature and then reduced (10mM DTT, 56°C, 2h) to alkane Kylation (25mM IAA, protected from light at room temperature, 40min), followed by adding Lys-C at an enzyme / protein ratio of 1:25 for enzymatic hydrolysis and reacting in a 37°C water bath for 16h.

[0025] 2) Edman degradation of protein enzymatic peptides: under alkaline conditions (pH = 8.0), 45 ° C React with the protein enzymatic peptide, the reaction time is 30 minutes. After the reaction mixed solution was lyophilized, 10 microliters to 1 milliliter of anhydrous trifluoroacetic acid was added and reacted at 45° C. for 10 minutes to cleave the N-terminal amino acid of the peptide from the peptide.

[0026] 3) Enrichment of protein C-term...

Embodiment 3

[0028] Such as figure 1 As indicated, protein C-terminal peptide enrichment was carried out as follows:

[0029] 1) Enzymatic hydrolysis of protein: The two extracted plasma proteins were dissolved in 50mM NaHCO3 (pH7.5), then heat denatured at 90°C for 20min, cooled to room temperature and then reduced (10mM DTT, 56°C, 2h) to alkane Kylation (25mM IAA, protected from light at room temperature, 40min), followed by adding Lys-C at an enzyme / protein ratio of 1:25 for enzymatic hydrolysis and reacting in a 37°C water bath for 16h.

[0030] 2) Edman degradation of protein enzymatic peptides: under alkaline conditions (pH = 8.0), 45 ° C React with the protein enzymatic peptide, the reaction time is 30 minutes. After the reaction mixed solution was lyophilized, 10 microliters to 1 milliliter of anhydrous trifluoroacetic acid was added and reacted at 45° C. for 10 minutes to cleave the N-terminal amino acid of the peptide from the peptide.

[0031] 3) Enrichment of protein C-term...

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Abstract

The invention relates to a protein C-terminal peptide enrichment method based on Edman degradation. According to the protein C-terminal peptide enrichment method based on Edman degradation, a protease used for enzyme digestion of peptide chains after lysine is used for enzymatic hydrolysis of a protein sample so as to provide the C terminal of an obtained protein non-C-terminal peptide with a lysine, wherein the protein C-terminal peptide generally contains no lysine; the peptide segments obtained via enzymatic hydrolysis is labeled with an amino reaction reagent with physicochemical property improving functional groups and isorhodanate functional groups; after labeling, Edman degradation is carried out under strong acidic conditions so as to cut the N-terminal first amino acid of the peptide segments off from the peptide segments; the C terminal lysine side chain amino groups of the protein non-C-terminal peptide are labeled with the physicochemical property improving functional groups, so that the physicochemical properties of the protein non-C-terminal peptide are obviously different from that of protein C-terminal peptide; selective absorption of the protein non-C-terminal peptide is carried out based on the different of the physicochemical properties of the protein non-C-terminal peptide and that of the protein C-terminal peptide so as to obtain the protein C-terminal peptide via enrichment.

Description

technical field [0001] The present invention relates to a protein C-terminal peptide enrichment method assisted by Edman degradation, specifically a method for enzymatically hydrolyzing a protein sample by using a protease that cuts after lysine, and then using Edman degradation reaction, Selectively label the physicochemical property-changing functional group on the amino group of the lysine side chain of the non-C-terminal peptide of the protein, and then selectively adsorb the non-C-terminal of the protein according to the difference in the physicochemical properties of the protein C-terminal peptide and the non-C-terminal peptide peptides, thereby enriching the C-terminal peptides of proteins. Background technique [0002] Protein terminal cleavage is a common and important modification, and the resulting terminal sequence has an important impact on the biological function of the molecule. For example, some protein precursors require proteolytic cleavage to remove the s...

Claims

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Application Information

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IPC IPC(8): G01N1/40
Inventor 张丽华单亦初陈玲凡张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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