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Protein C-terminal enriching method based on carboxypeptidase and strong cation exchange chromatography

A strong cation exchange, protein technology, applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., can solve the problems of low chemical reaction efficiency, cumbersome and time-consuming process, lack of specificity, etc., to promote efficient removal and enrichment. The effect of high collection efficiency and high carboxypeptidase digestion efficiency

Inactive Publication Date: 2017-11-24
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these enrichment methods have disadvantages such as low chemical reaction efficiency, uncontrollable side reactions, cumbersome and time-consuming process, and insufficient specificity.

Method used

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  • Protein C-terminal enriching method based on carboxypeptidase and strong cation exchange chromatography
  • Protein C-terminal enriching method based on carboxypeptidase and strong cation exchange chromatography
  • Protein C-terminal enriching method based on carboxypeptidase and strong cation exchange chromatography

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Such as figure 1 As shown, the protein sample first blocked the free carboxyl groups of the C-terminal and side chains at the protein level, and then digested the basic sites of the protein to produce intermediate peptides with basic amino acids at the carboxy-terminal, and ion-exchange chromatography was used to detect The digested products were fractionated to obtain multiple fractions, and finally the basic amino acids at the carboxy-terminal of the intermediate peptide were cut, and each fraction was separated by secondary ion exchange chromatography to exclude intermediate peptides with shifted retention times. Obtain C-terminal peptides of proteins

[0036] Take the peptide segment AVGNHLK ending in K as the sample, dissolve it in 50mM ammonium bicarbonate, and digest it with carboxypeptidase B. The amount of enzyme used is 1 / 5 of the sample mass, and the temperature is 37°C. After 30 minutes of enzymatic digestion, mass spectrometry analysis is carried out. ,Suc...

Embodiment 2

[0038] Take the peptide segment AVGNHLK ending in K as the sample, dissolve it in 50mM ammonium bicarbonate, and digest it with carboxypeptidase B. The amount of enzyme used is 1 / 10 of the sample mass, and the temperature is 37°C. After enzymatic digestion for 30 minutes, perform mass spectrometry analysis , the lysine at the carboxy-terminal of the same peptide was cleaved efficiently and selectively.

Embodiment 3

[0040] Take the peptide AVGNHLK ending in K as the sample, dissolve it in 50mM ammonium bicarbonate, and digest it with carboxypeptidase B, where the amount of enzyme used is 1 / 10 of the sample mass, and the temperature is 37°C. After enzymatic digestion for 45 minutes, perform mass spectrometry analysis , the lysine at the carboxy-terminal of the same peptide was cleaved efficiently and selectively.

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Abstract

The invention relates to a protein C-terminal enriching method based on a carboxypeptidase and strong cation exchange chromatography. The protein C-terminal enriching method comprises the steps of closing of the protein free carboxyl, enzyme digestion of protein alkaline loci, separation by adopting the ion exchange chromatography and cutting of peptide fragmental carboxyl terminal alkaline amino acid. A protein sample closes the free carboxyl at the tail end and a side chain of C at a protein level firstly, then enzyme digestion is conducted on the protein alkaline loci so as to generate a middle peptide fragment, and the carboxyl terminal is the alkaline amino acid; grading is conducted on an enzyme-digested product by adopting the ion exchange chromatography to obtain a plurality of fractions; and finally cutting is conducted on the alkaline amino acid of the carboxyl terminal of the middle peptide fragment, secondary ion exchange chromatographic separation is conducted on each friction so as to exclude the middle peptide fragment which shifts in the retention time, and thus the C terminal peptide fragment of the protein is obtained. The protein C-terminal enriching method has the advantages that the enzyme digestion efficiency, the removal efficiency and the enriching efficiency are high, multiple enzymes can be adopted to conduct enzyme digestion, and the coverage degree of identification of the C terminal is improved.

Description

technical field [0001] The invention relates to a protein C-terminal enrichment method, that is, a protein C-terminal enrichment method using carboxypeptidase and strong cation exchange chromatography, so as to realize high-efficiency and high-selectivity enrichment of protein C-terminal. Background technique [0002] The modification and cleavage of protein C-terminal is one of the common post-translational modifications in organisms, which is related to the process of protein activity, localization and complex formation. In addition, the degradation of proteins in vivo promotes the generation of new C-terminals, which are closely related to many biological processes and physiological functions such as apoptosis and chemokine processing. However, the protein components in biological samples are complex, and the dynamic distribution range of the content is wide, and the Bottom-up strategy further increases the complexity of the sample. Therefore, developing enrichment metho...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K1/18
CPCC07K1/18C12P21/06
Inventor 张丽华陈玲凡单亦初杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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