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Integrated protein C-terminal enrichment method

A protein enrichment technology, applied in the field of protein C-terminal enrichment, can solve the problems of difficult to control side reactions, cumbersome and time-consuming process, low reaction efficiency, etc., to reduce time and sample consumption, high reaction efficiency, and processing steps simple effect

Active Publication Date: 2014-06-25
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

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

Method used

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  • Integrated protein C-terminal enrichment method
  • Integrated protein C-terminal enrichment method
  • Integrated protein C-terminal enrichment method

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Embodiment 1

[0029] like figure 1 As shown, the protein is enzymatically hydrolyzed by Lys-C to produce peptides with side chain amino groups except the C-terminus. The enzymatic hydrolyzed products are passed into the C18 trapping column, labeled on the column and desalted, and then eluted to TiO 2 The peptides marked with phosphate groups are removed by adsorption on the trap column to obtain C-terminal peptides.

[0030] Taking β-casein as a sample, Lys-C was used for enzymatic hydrolysis, wherein the amount of enzyme was 1 / 100 (w / w) of the sample mass, the temperature was 37 °C, pH=8, and the enzymatic hydrolysis was performed for 12 h. The enzymatic hydrolysate was pushed into a C18 trap column (0.15mm i.d×50mm), and 2% ACN was passed through to remove the salt. A dimethylation labeling solution was introduced, wherein the labeling solution consisted of 500 μL 1% acetic acid + 5 μL 4% formalin solution + 5 μL 0.6M sodium cyanoborohydride solution (1% acetic acid), flow rate 5 μL / min,...

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Abstract

The present invention relates to an integrated protein C-terminal enrichment method, which comprises: selective dimethylation labeling, DL-glyceraldehyde-3-phosphate labeling and affinity removing with titania. According to the method, an enzymolysis product of a protein sample is subjected to selective N-terminal amino dimethylation labeling on a reverse phase trapping column under an acid condition, the side chain amino of the peptide segment is labeled on a DL-glyceraldehyde-3-phosphate column, and finally a TiO2 material is adopted to carry out affinity removing to remove the phosphate-labeled peptide segment so as to obtain the protein C-terminal, wherein sample transfer and desalination lyophilization are not required to be performed during the process. The method has advantages of high labeling efficiency, high selectivity, simple treatment steps, time saving, sample consumption saving, and sample loss and contamination reducing. In addition, the method provides good compatibility with the chromatography-mass spectrometry separation and identification platform.

Description

technical field [0001] The invention relates to a protein C-terminal enrichment method, which integrates the process to realize rapid and efficient enrichment of protein C-terminal. Background technique [0002] Bottom-up strategies are currently the most widely used in proteome separation assays. However, this strategy separates and identifies tens of thousands of proteins into hundreds of thousands or even millions of peptide fragments, which not only poses a high challenge to chromatographic separation, but also affects the accuracy of mass spectrometry identification. Therefore, it is of great significance to develop new methods for the separation and identification of characteristic peptides to improve the ability of protein separation and identification. According to statistical analysis, the C-terminal region of the protein is highly sequence specific, and the four amino acid sequences at the end of the protein are unique to 74% to 97% of the proteins [1], so the seq...

Claims

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Application Information

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IPC IPC(8): G01N1/40
Inventor 张丽华陈玲凡袁辉明周愿杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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