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Method for purifying tyrosine phosphopeptide

A technology of tyrosine phosphorylation and tyrosine phosphopeptide, which is applied in the purification field of tyrosine phosphorylated peptide, can solve the problems of harsh solvent and temperature requirements, limited reagent price, difficult protein identification, etc., and achieves high sensitivity, Highly selective, highly specific effects

Inactive Publication Date: 2013-04-24
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

Although the efficiency of enriching P-Tyr phosphopeptides at the peptide level is high, the low sequence coverage leads to difficulties in protein identification, harsh solvent and temperature requirements, and expensive reagents limit its wide application.

Method used

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  • Method for purifying tyrosine phosphopeptide
  • Method for purifying tyrosine phosphopeptide
  • Method for purifying tyrosine phosphopeptide

Examples

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Effect test

preparation example Construction

[0027] Preparation of sample solution: 1 mg each of α-casein and β-casein were dissolved in 1 mL of 50 mM ammonium bicarbonate solution (pH 8.0), according to the mass ratio of trypsin to 1:40 (w / w) Trypsin was added for enzymolysis reaction, the reaction time was 16 hours, and the enzymolysis temperature was controlled at 37°C. The obtained proteolysis solution was stored in a -30°C refrigerator for future use. Dissolve 1mg of protein bovine serum albumin in 1mL of reducing solution ((pH 8.0, 8M urea, 50mM ammonium bicarbonate solution), leave it at room temperature for 4h, then add DTT solution to the centrifuge tube containing inactivated protein , placed in a water bath at 37°C for 2 hours. Take the solution out of the water bath, add IAA solution to each tube of solution, and let it stand in the dark at room temperature for 30 minutes. After the above treatment, the protein is completely denatured, the disulfide bond is opened, and the sulfhydryl group is blocked. Then t...

Embodiment 1

[0033] Example 1. β-elimination and Michael nucleophilic addition reaction method for purification of tyrosine phosphopeptides

[0034] (1) The enzymatic hydrolysis product of the standard phosphorylated protein β-casein and pY are mixed at a molar ratio of 1 pmol: 1 pmol.

[0035] (2) Use Ti 4+ -IMAC monodisperse microspheres enrich and purify phosphopeptides. Disperse the above-prepared samples in 50% ACN, 6% TFA, and 200 mM NaCl on Ti 4+ - The suspension of IMAC monodisperse microspheres (10mg / mL) was mixed according to the mass ratio of sample and microspheres at 1:10, incubated with shaking at room temperature for 30min, then centrifuged at 30000g at high speed, discarded the supernatant, and added an equal volume of Aqueous solution containing 200mM NaCl, 50% ACN, 6% TFA, shaking to clean Ti 4+ -IMAC monodisperse microspheres for 15min, then centrifuge at 30,000g at high speed, discard the supernatant, then add twice the volume of an aqueous solution containing 0.1% T...

Embodiment 2

[0042] Example 2. The difference from Example 1 is that in the complex sample system, the moles of pY, α-casein, β-casein and BSA are 1:10:10:10 and 1:100:100:100. Depend on Figure 4 It can be seen that in the complex sample system, the molar ratio of pY, α-casein, β-casein and BSA is 1:10:10:10 and 1:100:100:100, the untreated sample is very complex, but after β-elimination The sample treated with the Michael nucleophilic addition reaction method left only pY, and it was very clean with a very good signal-to-noise ratio. This shows that the method of β-elimination / Michael nucleophilic addition reaction has very high specificity for purifying P-Tyr phosphopeptide.

[0043] purification

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Abstract

The invention discloses application of a beta-elimination and Michael nucleophilic addition method in study of phosphoproteomics, in particular a method for purifying tyrosine phosphopeptide. The method comprises the following steps of: eliminating phosphate modification groups on non-tyrosine phosphopeptide in protein hydrolysate by using beta-elimination and Michael nucleophilic addition; and enriching the tyrosine phosphopeptide which is not subjected to the elimination reaction by using a phosphoeptide enriching material. By utilizing the high specificity of the beta-elimination and Michael nucleophilic addition, the tyrosine phosphopeptide is purified from a complicated protein hydrolysate sample in high selectivity.

Description

technical field [0001] The present invention is a purification method of tyrosine phosphorylated peptide based on β-elimination and Michael nucleophilic addition reaction, and is the application of β-elimination and Michael nucleophilic addition method in phosphorylated proteome, β-elimination The method of nucleophilic addition reaction with Michael shows high specificity, high selectivity and high sensitivity to tyrosine phosphorylated peptides. β-elimination and Michael nucleophilic addition reactions can realize the purification of tyrosine phosphopeptides, which can be applied to the phosphoproteome of protein post-translational modification. Eliminate the phosphate groups on serine and threonine by β-elimination and Michael nucleophilic addition reaction methods, and then use phosphopeptide enrichment materials to purify tyrosine phosphorylated peptides from complex proteolysis samples, combined with biological mass spectrometry Perform large-scale identification of tyr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14
Inventor 邹汉法韩广辉叶明亮王春丽
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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