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Proteome sample pretreatment method based on novel nanometer composite material, and applications thereof

A nanocomposite material and sample pretreatment technology, applied in the field of preparation of nano-gold modified graphene oxide nanocomposites, can solve the problems of low-abundance protein masking, long running time, cumbersome operation, etc., and achieve good reproducibility, Reduced complexity, particle stabilization effects

Active Publication Date: 2015-06-17
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the liquid isoelectric focusing method can only classify the samples according to the limited isoelectric point interval according to the existing isoelectric point film.
Therefore, the fraction of high-abundance proteins still has the problem of masking low-abundance proteins
The multidimensional liquid chromatography method is not only cumbersome to operate, but also takes a long time to run, and it is difficult to avoid removing high-abundance proteins while causing co-elution of low-abundance proteins.

Method used

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  • Proteome sample pretreatment method based on novel nanometer composite material, and applications thereof
  • Proteome sample pretreatment method based on novel nanometer composite material, and applications thereof
  • Proteome sample pretreatment method based on novel nanometer composite material, and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Material Characterization

[0023] The prepared polyethyleneimine-coupled nano-gold modified graphene oxide nanocomposite particles were characterized by transmission electron microscopy as follows: figure 1 As shown, according to this figure, it can be seen that the nano-gold particles are uniformly dispersed in the graphene layer, indicating that the modified polyethyleneimine on the graphene layer successfully reduces the chloroauric acid solution, generates nano-gold particles on its surface, and the material Has very good dispersibility.

[0024] 2. Sample handling process

[0025] The prepared nanocomposite particles were incubated with plasma protein samples in three phosphate buffer solutions with pH values ​​of 4.0, 7.4, and 9.0 for 0.5 h, and the incubation temperature was 25°C. After the particles are centrifuged to remove the supernatant, the phosphate buffer with the same pH value as the incubation solution is used for washing, and the supernatant is r...

Embodiment 2

[0028] 1. Enzymatic hydrolysis of proteins and nanocomposites

[0029] The compound of the obtained nano-composite material and protein was heated and denatured, and then trypsinized respectively. The specific process is as follows: Denature the sample at 90°C for 20 minutes, add a certain amount of dithiothreitol (DTT, add 8 μmol of dithiothreitol solution per mg of protein), and react at 56°C for 1.5 hours to achieve protein reduction. Process, then add iodoacetamide solution (IAA, 1 μmol DTT adds 2.5 μmol IAA) in proportion, react 40min under dark conditions to carry out alkylation to protein, finally add trypsin (the mass ratio of trypsin and protein is 1: 25), react at 37°C for 16h.

[0030] 2. Ion exchange-reversed phase tandem liquid mass analysis and data processing

[0031]After centrifuging the material after the above enzymatic hydrolysis, the supernatant was collected and analyzed by ion exchange-reverse phase tandem liquid mass (SCX-RPLC-MS / MS). Three mobile ph...

Embodiment 3

[0036] The prepared nanocomposite material was incubated with mouse brain tissue protein samples in citric acid buffer solution of pH 3.0, sodium acetate buffer solution of pH 9.0 and phosphate buffer solution of pH 7.0, and the tissue samples were processed.

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Abstract

The present invention relates to a new method for using a novel nanometer composite material for protein sample treatment. According to the present invention, with the application of the material in the protein sample pretreatment, the abundance of the high-abundance proteins can be effectively reduced, the complexity of the sample can be reduced, and the more low-abundance proteins in the blood plasma can be identified; and compared with the traditional protein sample pretreatment method, the protein sample pretreatment method of the present invention has characteristics of convenience, easy performing, low cost and the like.

Description

technical field [0001] The present invention relates to a protein sample pretreatment method, specifically a preparation of a nano-gold modified graphene oxide nanocomposite based on polyethylene glycol-modified polyethyleneimine coupling and its application in protein sample pretreatment . Background technique [0002] The composition of proteins in biological samples is extremely complex, and the dynamic range is relatively wide, with large concentration differences (large abundance differences). For example, the content of the top 10 high-abundance proteins in plasma samples accounts for 90% of the total protein content, and the top 22 proteins It accounts for 99%, but the remaining thousands of proteins account for only 1%. The existence of high-abundance proteins will cover up low-abundance proteins, thereby interfering with the identification of low-abundance proteins, and these low-abundance proteins often have potential Biological significance, may play an important...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/88
Inventor 张丽华邓楠江波陈远波吴琪杨开广梁振张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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