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Method for enriching and detecting phosphorylated proteins

A phosphorylated protein, non-phosphorylated technology, applied in the fields of material analytical chemistry and organic chemistry, can solve the problems of difficult separation of phosphorylated proteins, gaps in phosphorylation information, etc., and achieve high separation efficiency, small sample loss, and high selectivity. Effect

Inactive Publication Date: 2017-05-31
WUHAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, due to the difficulty in separating intact phosphorylated proteins, the research on phosphoproteomics is mainly based on the "bottom-up" strategy, that is, the protein is first indiscriminately hydrolyzed into polypeptide chains, and the phosphorylation sites are identified at the peptide level. Points, and then perform database search, reasoning and splicing to obtain complete phosphorylated protein information. Although this method is simple and feasible, there is a big gap between the phosphorylated information at the protein level and the real information in actual biological samples.

Method used

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  • Method for enriching and detecting phosphorylated proteins
  • Method for enriching and detecting phosphorylated proteins
  • Method for enriching and detecting phosphorylated proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Preparation of enriched material

[0048] Two-component copolymer structures such as figure 1shown, where X=0.01-0.5. Taking X=0.2 as an example, in a 25ml three-necked flask, add 0.4mmol of isopropylacrylamide and 0.1mmol of thiourea functional monomer successively, the substance molar ratio is 4:1, and at the same time add 6mL of ultrapure water and 6mL of DMF as solvent Introduce nitrogen under stirring, after the monomer is fully dissolved, add catalyst CuBr 0.032g and bipyridine ligand 0.16mL under nitrogen protection, then the reaction system is evacuated and filled with nitrogen to remove residual oxygen in the reaction system; Immerse the brominated substrate into the prepared reaction solution; control the temperature of the flask at 70-90 ° C and let it stand for 12-36 hours; after the reaction, use N,N'-dimethylformamide (DMF) and Deionized water (H 2 O) Wash the polymer graft surface in turn to obtain an enriched material. The thickness of the two-compone...

Embodiment 2

[0051] Structure and Synthesis of Functional Monomers

[0052] In order to prepare the above-mentioned polymer, it is necessary to synthesize a series of thiourea functional monomers, and the synthetic methods of their specific implementation are similar, and the synthetic steps are as follows. image 3 shown.

[0053] Taking the synthesis of a thiourea monomer whose terminal group R is a carboxyl group as an example, at room temperature, dissolve 0.582g (6mmol) in 30-50mL of anhydrous acetone, and under stirring, add 0.453g (5mmol) of acryloyl chloride dropwise It was added dropwise to the above solution, and the reaction was continued for 12 hours. After the reaction, centrifuge (4500r / min, 5min), and take the supernatant for use. At room temperature, 0.685g (6mmol) of p-aminobenzoic acid was dissolved in 30mL of anhydrous acetone, 2 to 4mL of ultrapure water was added, and the standby clear liquid was added dropwise to the above solution under stirring conditions, and the...

Embodiment 3

[0060] The adsorption behavior of α-casein and bovine serum albumin on the surface of the enriched two-component copolymer was evaluated by the method of QCM-D adsorption capacity determination, taking the thiourea monomer whose R-terminal group is a carboxyl group as an example. The polymer was grafted to the surface of the QCM-D chip as described in Example 1, and α-casein and bovine serum albumin were adsorbed with 10% acetonitrile + 90% deionized water as the carrier liquid under the temperature control of 20 °C. experiment. Figure 4 The different adsorption behaviors of α-casein and bovine serum albumin on the surface of the two-component copolymer were shown, which fully demonstrated the ability of this two-component copolymer to specifically adsorb phosphorylated proteins.

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Abstract

The invention relates to the field of chemical analysis of materials and organic chemistry, and provides a method for enriching and detecting phosphorylated proteins. The method comprises the following steps: directly mixing a protein mixture, which is obtained by mixing phosphorylated proteins and non-phosphorylated proteins, with an enriching material, separating and enriching the non-phosphorylated proteins and the phosphorylated proteins by adopting a dispersive solid phase extraction mode, and carrying out chromatographic analysis on a sample, wherein the enriching material comprises a substrate and a double-component copolymer layer formed on the surface of the substrate; the double-component copolymer layer is 50 to 100 nm in thickness. According to the method, the enriching material and the dispersive solid phase extraction mode are organically combined, so that high-selectivity, high-repetitiveness and high-flux enrichment of the phosphorylated proteins in a complicated mixture can be realized.

Description

technical field [0001] The invention relates to the fields of material analysis chemistry and organic chemistry, in particular to a method for enriching and detecting phosphorylated proteins. Background technique [0002] Phosphorylation of proteins is a reversible post-translational modification that plays an extremely important role in cell proliferation, differentiation, signal transduction, transcription and translation, protein complex formation, and protein degradation. Therefore, phosphorylated proteins have become a hot issue in proteomics research. Phosphorylated proteins in natural samples are difficult to be directly detected by the currently commonly used mass spectrometry. In order to solve this problem and improve the signal response of mass spectrometry to phosphorylated proteins, phosphorylated proteins must be separated and enriched, which has become an important part of phosphorylated proteomics research. At present, due to the difficulty of separating in...

Claims

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Application Information

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IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8831
Inventor 卿光焱孙涛垒陆琦
Owner WUHAN UNIV OF TECH
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