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Phosphopeptide enriching material and preparation method and application thereof

A phosphorylated peptide and enrichment technology, which is applied in the field of material analytical chemistry and organic chemistry, can solve the problem of difficult elution of multi-phosphorylated peptides, inability to enrich phosphorylated peptides, non-specific adsorption of acidic peptide chains with basic residues Phosphorylated peptides are difficult to retain and other problems, so as to achieve the effects of easy repeatability, selective enrichment, and improved detection limit and sensitivity

Inactive Publication Date: 2017-05-31
WUHAN UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods have the problems of difficult elution of multi-phosphorylated peptides, non-specific adsorption of acidic peptide chains, or difficulty in retention of phosphorylated peptides with basic residues, so the purpose of large-scale enrichment of phosphorylated peptides in complex biological samples cannot be achieved.

Method used

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  • Phosphopeptide enriching material and preparation method and application thereof
  • Phosphopeptide enriching material and preparation method and application thereof
  • Phosphopeptide enriching material and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Preparation of phosphorylated peptide enrichment material

[0049] Two-component polymer structure such as figure 1 Shown, where X = 0.01-0.5. Taking X=0.2 as an example, add 0.4mmol of isopropylacrylamide and 0.1mmol of thiourea functional monomer into a 25ml three-necked flask in turn, the molar ratio of the substances is 4:1, and add 6mL of ultrapure water and 6mL of DMF as solvents at the same time ; Feed 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—nitrogen-filled to remove residual oxygen in the reaction system; Immerse the brominated substrate into the prepared reaction solution; control the temperature of the flask at 60-80°C and let it stand for 4-24 hours; after the reaction, use N,N'-dimethylformamide (DMF) and Deionized water (H 2 O) washing the surface of the polymer graft in sequence to obtain a phosphorylated peptide-...

Embodiment 2

[0052] Structure and Synthetic Method of Functional Monomer

[0053] In order to prepare the above-mentioned two-component copolymer, a series of thiourea functional monomers need to be synthesized, and the synthetic method of their specific implementation is similar, and the synthetic steps are as follows: image 3 shown.

[0054] Taking the synthesis of thiourea monomer whose end group R is carboxyl as an example, dissolve 0.582g (6mmol) in 30-50mL of anhydrous acetone at room temperature, and add 0.453g (5mmol) of acryloyl chloride dropwise under stirring conditions. Add it dropwise to the above solution, and continue the reaction for 12 hours. Centrifuge (4500r / min, 5min) after the reaction, and take the supernatant for later use. At room temperature, dissolve 0.685g (6mmol) of p-aminobenzoic acid in 30mL of anhydrous acetone, add 2-4mL of ultrapure water, and add the spare clear solution dropwise to the above solution under stirring, and continue the reaction for 12 Ho...

Embodiment 3

[0061] The specific adsorption of phosphorylated peptides on the surface of two-component copolymers was evaluated by the method of QCM-D adsorption measurement, taking the thiourea monomer whose R terminal group is carboxyl group as an example. The two-component copolymer was grafted onto the surface of the QCM-D chip as described in Example 1, and the non-phosphorylated Peptides and mono-, di-, and tri-phosphorylated peptides were subjected to adsorption experiments, respectively. Figure 4 The adsorption curves of different peptide chains on the surface of the two-component copolymer are shown, which fully demonstrates the excellent specific adsorption of phosphorylated peptides of this type of polymer and the ability to effectively distinguish the number of phosphorylated sites. The field of enrichment and separation has very broad application prospects. 000000

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Abstract

The invention relates to the field of material analysis chemistry and organic chemistry. The invention provides a phosphopeptide enriching material and a preparation method and application thereof. The phosphopeptide enriching material comprises a substrate and a double-ingredient copolymer layer formed on the surface of the substrate, wherein the thickness of the double-ingredient copolymer layer is 10 to 80nm. The surface initiation-atom transfer free radical polyreaction mechanism is utilized; the double-ingredient copolymers are grafted on the surface of the substrate. The material and column solid phase extraction mode or dispersion solid phase extraction mode are organically combined; the enriching with high selectivity, high repetitiveness and high flux of phosphopeptide in a complicated mixture can be realized, so that the selective enriching of the multi-phosphopeptide and mono-phosphopeptide can be realized; the phosphorylated protein identification number can be obviously improved. Therefore the phosphopeptide enriching material is hopeful to be widely applied to the aspects of phosphopeptide enriching, further large-scale separation of phosphorylated protein and the like.

Description

technical field [0001] The invention relates to the fields of material analytical chemistry and organic chemistry, in particular to a phosphorylated peptide enrichment material and its preparation method and application. Background technique [0002] Protein phosphorylation is one of the most common and important post-translational modification methods in organisms, so the study of phosphoproteomics has attracted the attention of many scientists around the world. However, because the absolute content of phosphorylated proteins in organisms is very low, the current technical strategy for phosphoproteomics research is mainly after enrichment at the protein or peptide level. Existing phosphopeptide enrichment methods mainly include: immobilized metal ion affinity chromatography, metal oxide method, ion exchange chromatography, and the combination of the above techniques. However, these methods have the problems of difficult elution of multi-phosphorylated peptides, non-specifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08F220/54C08F220/60C07K1/22
CPCC07K1/22C08F220/54C08F220/606
Inventor 孙涛垒梁鑫淼卿光焱李秀玲陆琦姜舸
Owner WUHAN UNIV OF TECH
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