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Chemical labeling and mass spectrometry-based structure analysis method for protein and material interaction

A technology of chemical labeling and analysis method, applied in the field of protein-material interaction structure analysis based on chemical labeling and mass spectrometry, can solve the problems of protein structure change, affecting protein electrostatic interaction, loss of activity, etc. Effects of structure and activity

Pending Publication Date: 2019-12-24
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in order to ensure high labeling efficiency, some covalent chemical labeling methods require the use of highly reactive amidation reagents, which neutralize the positive charge of the lysine side chain and affect the electrostatic interaction between proteins, resulting in labeling Changes in protein structure and loss of activity after

Method used

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  • Chemical labeling and mass spectrometry-based structure analysis method for protein and material interaction
  • Chemical labeling and mass spectrometry-based structure analysis method for protein and material interaction
  • Chemical labeling and mass spectrometry-based structure analysis method for protein and material interaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Structural analysis of protein-material interaction based on mass spectrometry:

[0053] 1. Prepare two groups of photosystem II proteins with a concentration of 0.01 μg / μL for the control group and the experimental group. Add iron oxide to the protein and materials in the experimental group at a ratio of 1:100, and incubate for 10 minutes at the same time;

[0054] 2. Add formaldehyde with a final concentration of 0.01mmol / L and sodium cyanoborohydride with a final concentration of 0.01mmol / L respectively, and react at 4°C for 120min;

[0055] 3. Add NH with a final concentration of 1mmol / L 4 HCO 3 ;

[0056] 4. Replace the solution obtained in the above step 3 with 8M urea, boil it, add chymotrypsin, the mass ratio of protein to enzyme is 50:1, and enzymolyze overnight at 25°C;

[0057] 5. The solution obtained in the above step 4 is acidified, and the chromatographic separation and the detection of mass spectrometry are carried out;

[0058] 6. Perform a database...

Embodiment 2

[0065] Structural analysis of protein-material interaction based on mass spectrometry:

[0066] 1. Prepare two groups of photosystem II proteins with a concentration of 0.1 μg / μL for the control group and the experimental group. Add iron oxide to the protein and materials in the experimental group at a ratio of 1:20, and incubate for 30 minutes at the same time;

[0067] 2. Add formaldehyde with a final concentration of 2mmol / L and sodium cyanoborohydride with a final concentration of 2mmol / L respectively, and react for 30min at room temperature;

[0068] 3. Add CH with a final concentration of 50mmol / L 3 COONH 4 ;

[0069] 4. Replace the solution obtained in the above step 3 with 6M guanidine hydrochloride, boil it, add chymotrypsin, the mass ratio of protein to enzyme is 25:1, and enzymolyze at 30°C overnight;

[0070] 5. The solution obtained in the above step 4 is acidified, and the chromatographic separation and the detection of mass spectrometry are carried out;

[0...

Embodiment 3

[0078] Structural analysis of protein-material interaction based on mass spectrometry:

[0079] 1. Prepare two groups of photosystem II proteins with a concentration of 10 μg / μL for the control group and the experimental group. Add iron oxide to the protein and materials in the experimental group at a ratio of 1:10, and incubate for 60 minutes at the same time;

[0080] 2. Add formaldehyde with a final concentration of 100mmol / L and sodium cyanoborohydride with a final concentration of 100mmol / L respectively, and react at 40°C for 5 minutes;

[0081] 3. Add NH with a final concentration of 1mmol / L 4 HCO 3 ;

[0082] 4. Replace the solution obtained in the above step 3 with 6M urea, boil it, add trypsin, the mass ratio of protein to enzyme is 25:1, and enzymolyze overnight at 37°C;

[0083] 5. The solution obtained in the above step 4 is acidified, and the chromatographic separation and the detection of mass spectrometry are carried out;

[0084] 6. Perform a database searc...

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Abstract

The invention relates to a new structure analysis method for researching protein and material interaction based on chemical labeling and mass spectrometry technologies. The method is characterized inthat firstly proteins and protein-material complexes are chemically labeled, then denaturation, enzymatic hydrolysis, chromatography-mass spectrometry analysis and database retrieval of the proteins are carried out, and the change conditions of the chemical labeling efficiency of specific amino acid sites on the proteins before and after materials are added are compared, so that a region with theobviously reduced labeling efficiency is namely a protein-material interaction region and a region with the obviously improved labeling efficiency is namely a protein conformation change region afterthe materials are combined. The protein-material interaction analysis method has the advantages of being rapid and simple, stable and efficient, high in sensitivity and capable of realizing high-throughput dynamic analysis, the physiological structures and activities of the proteins are still kept in the labeling process, and an analysis result is not limited by conditions such as the molecular weight, the purity and the like of the proteins.

Description

technical field [0001] The invention belongs to the research field of using proteomics analysis technology to study the interaction between proteins and materials, and specifically relates to a new method for structural analysis based on chemical labeling and mass spectrometry technology, to study the conformational change regions of proteins after the interaction between proteins and materials, and the protein and materials. The interaction area of ​​the material. Background technique [0002] At present, nanomaterials have been used in life sciences, medicine and other fields as tools for diagnosis and treatment, and some nanomedicines have also entered the stage of clinical trials or clinical applications. When these materials enter the body, the materials will interact with the proteins in the body, causing a series of biological reactions, such as how these materials bind to different proteins, and how the conformation of proteins will change after binding materials. c...

Claims

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Application Information

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IPC IPC(8): G01N30/06G01N30/88G01N30/72
CPCG01N30/06G01N30/72G01N30/88
Inventor 王方军贺敏
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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