Applications of ZrO2 in the process for concentrating and purifying phosphorylated peptides

A technique for phosphorylated peptides and enrichment, applied in the field of enrichment and purification of phosphorylated peptides in the phosphorylated proteome

Inactive Publication Date: 2008-10-15
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
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Problems solved by technology

But using ZrO 2 The specific interaction between nanomaterials and phosphorylated peptides to enrich and purify phosphorylated peptides has not been reported, and conventional commercial immobilized metal ion affinity chromatography (IMAC), TiO 2 , ZrO 2 Microcolumn, Al(OH) 3 Compared with other enrichment methods, ZrO 2 Nanomaterials Purification and Enrichment of Phosphopeptides Show Higher Specificity, Selectivity, Binding Capacity and Sensitivity

Method used

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  • Applications of ZrO2 in the process for concentrating and purifying phosphorylated peptides
  • Applications of ZrO2 in the process for concentrating and purifying phosphorylated peptides
  • Applications of ZrO2 in the process for concentrating and purifying phosphorylated peptides

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preparation example Construction

[0028] ZrO 2 The preparation of nanomaterials is obtained by co-precipitation method. The method is to use ZrOCl 2 The soluble zirconium salt and the precipitating agent ammonia are co-precipitated in the solution, and then calcined at high temperature to obtain ZrO 2 nanomaterials. This preparation of ZrO 2 The method of nanomaterials is simple, but the obtained nanomaterials have the problem of agglomeration. This can be obtained from figure 1 It can be seen in the scanning electron microscope image. Obtained ZrO 2 The size of nanoparticles is between 20-30nm.

[0029] ZrO 2 Synthesis of Nanomaterials

[0030] At room temperature, maintain pH = 10.0 under slow stirring to ZrOCl 2 .8H 2 O slowly add 25% NH 3 .H 2 O, get Zr(OH) 4 The precipitate was washed with double distilled water until the chloride ions were completely removed. Washed Zr(OH) 4 Drying at 110°C for 12 hours followed by calcination at 550°C for 4 hours yielded ZrO 2 Nanoparticles, scanning el...

Embodiment 1

[0039] 1. Preparation of sample solution: 1 mg of α-casein and β-casein were respectively dissolved in 1 mL of 50 mM ammonium bicarbonate solution (pH 8.2), according to the mass ratio with trypsin 1: 40 (w / w ) ratio to add trypsin for enzymolysis reaction, the reaction time is 16h, and the enzymolysis temperature is controlled at 37°C. The obtained proteolysis solution was stored in a -30°C refrigerator for future use.

[0040] 1mg of protein bovine serum albumin and ovalbumin were respectively dissolved in 1mL of reducing solution ((pH 8.2, 8M urea, 50mM ammonium bicarbonate solution), placed at room temperature for 4h, and then centrifuged into the centrifuge containing the inactivated protein Add DTT solution to the tube, and place it in a water bath at 37°C for 2 hours. Take the solution out of the water bath, add IAA solution to each tube solution, and let it stand in the dark at room temperature for 30 minutes. After the above treatment, the protein is completely denatu...

Embodiment 2

[0052] The difference from Example 1 is that

[0053] 2. ZrO 2 Nanomaterial-specific enrichment and purification of phosphorylated peptides

[0054] (1) β-casein, α-casein and Ovalbumin, which are hydrolyzed products of standard phosphorylated proteins, were respectively prepared in 10% HAC (pH=2-3) as the loading solution.

[0055] (5) Add 10 μL of 25% NH 3 .H 2 O Ultrasonic cleaning of ZrO 2 The nanoparticles were centrifuged at 30,000 rpm for 30 minutes, and the supernatant was collected.

[0056] Analysis results: Phosphorylated peptides from phosphorylated proteins α-casein, β-casein and ovalbumin digested by ZrO 2 Nanomaterials capture, but non-phosphorylated peptides are eluted, indicating that ZrO 2 Nanomaterials can specifically enrich and purify phosphorylated peptides. The phosphorylated peptides from the enzymatic hydrolysis products of phosphorylated protein β-casein and non-phosphorylated protein bovine serum albumin were treated with ZrO 2 Captured by na...

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Abstract

The invention relates to the enrichment and purification of phosphopeptide in phosphoproteome, in particular to the application of ZrO2 in the enrichment and purification of phosphopeptide. ZrO2 nano material shows high specificity, selectivity, binding capacity and sensitivity at a phosphopeptide section; meanwhile, the affinity of the ZrO2 nano material to the specificity of phosphopeptide ensures that the material can be used in phosphoproteome during protein post-translational modification. Due to the specificity combination of ZrO2 with the phosphopeptide section in complex proteolysis sample, large-scale identification of phosphorylated protein and determination of phosphorylation sites can be realized through combining with MALDI-TOF MS and nano-LC MS/MS.

Description

technical field [0001] The present invention relates to the enrichment and purification of phosphopeptides in the phosphoproteome, specifically ZrO 2 In the process of enriching and purifying phosphorylated peptides, it is a ZrO-based 2 A method for the highly specific and selective enrichment and purification of phosphopeptides by nanomaterials. Background technique [0002] Post-translational protein modification is a hot topic in proteome research. Protein phosphorylation is the most common and important post-translational modification of proteins. Protein phosphorylation and dephosphorylation regulate almost the entire process of human life activities, including cell proliferation, development and differentiation, neural activity, muscle contraction, and metabolism. , tumorigenesis, etc., protein phosphorylation is still the main transmission mode of signals known so far. [0003] Traditional methods for protein phosphorylation analysis include radioisotope labeling, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
Inventor 邹汉法周厚江叶明亮徐松云
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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