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Method for extracting exosome in urine and method for analyzing proteomics and phosphorylation proteomics of exosome

A technology of exosomes and urine, which is applied in the fields of biotechnology and analytical chemistry, can solve the problems of cumbersome steps, long extraction time, and low extraction efficiency, and achieve the effects of simple operation, fast extraction speed, and improved extraction efficiency

Inactive Publication Date: 2019-11-22
ACADEMY OF MILITARY MEDICAL SCI
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Problems solved by technology

At present, the commonly used exosome extraction methods include ultracentrifugation, precipitation kit method, immunoaffinity method and microfluidic chip method, etc. These methods have the disadvantages of long extraction time, cumbersome steps, and low extraction efficiency.

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  • Method for extracting exosome in urine and method for analyzing proteomics and phosphorylation proteomics of exosome

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Embodiment 1

[0047] Example 1. Preparation of magnetic nanomaterials with surface chelated metal ions and use for enrichment of urine exosomes and proteomics and phosphoproteomics analysis

[0048] 1. Preparation of magnetic nanomaterials with surface chelated metal ions

[0049] according to figure 1 The schematic diagram of the synthetic route shown in the preparation of the magnetic nanomaterial surface chelated metal ions, the specific steps are as follows:

[0050] Step 1, preparation of graphene oxide modified magnetic nanoparticles

[0051] Weigh 30 mg of graphene oxide, disperse it in 30 mL of ethylene glycol, and ultrasonicate for 2 hours with a power of 100 W until the graphene oxide is uniformly dispersed. Add 160 mg of ferric chloride and 30 mg of trisodium citrate, continue to sonicate until completely dissolved, add 700 mg of anhydrous sodium acetate, stir mechanically at room temperature for 30 min, and transfer the resulting homogeneous solution to a Teflon-lined stainles...

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Abstract

The invention discloses a method for extracting an exosome in urine and a method for analyzing proteomics and phosphorylation proteomics of the exosome. The invention firstly provides a magnetic nanomaterial with metal ions chelated on the surface. The magnetic nano material is prepared according to the steps of modifying magnetic nano particles on the surface of graphene oxide to obtain a product 1; coating the surface of the product 1 with a silicon dioxide layer to obtain a product 2; modifying the surface of the silicon dioxide layer of the product 2 with a silane coupling agent with amino to obtain a product 3; modifying the surface of the product 3 with a metal chelating agent to obtain a product 4; and chelating metal ions on the surface of the product 4. The magnetic nano materialcan be used for extracting the exosome in urine, and can be used for proteomics analysis or phosphorylation proteomics analysis of the exosome. According to the invention, phosphate radicals exposedon the surface of a membrane phospholipid bilayer of the exosome are combined with the metal ions through a double-coordination chemical effect, the high-specificity extraction of the urine exosome isrealized by combining an electrostatic adsorption effect at the same time, the extraction speed is high, and the recovery rate is high.

Description

technical field [0001] The invention relates to the extraction of exosomes in urine and its proteomics and phosphorylated proteomics analysis methods, and relates to the fields of biotechnology and analytical chemistry. Background technique [0002] Phosphorylation is an important post-translational modification of proteins, and phosphorylated proteins are involved in many important physiological processes, such as cell apoptosis, cell proliferation, and cell migration. Changes in the phosphorylation level of peptides are closely related to the occurrence and development of many diseases, and have potential application prospects as disease biomarkers. However, due to the unstable state of protein phosphorylation and the high abundance of phosphatases in body fluids, it brings great difficulties to the detection of phosphorylated proteins and phosphorylated peptides, thus limiting the use of phosphorylated proteins in the early diagnosis of diseases. Applications. Exosomes ...

Claims

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Application Information

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IPC IPC(8): G01N30/88G01N30/08G01N30/14
CPCG01N30/08G01N30/14G01N30/88G01N2030/8831
Inventor 秦伟捷焦丰龙高方园
Owner ACADEMY OF MILITARY MEDICAL SCI
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