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Protein analysis method based on hydrophobic amino acid specific protease

A technology of hydrophobic amino acid and analysis method, which is applied in the field of protein analysis based on hydrophobic amino acid-specific protease, and achieves the effect of improving the accuracy and application range, and increasing the number of identifications

Inactive Publication Date: 2019-06-04
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

At present, there is no report on the application of proteolytic enzymes specific for hydrophobic amino acids for enzymatic hydrolysis and proteomic analysis of standard proteins or complex protein samples

Method used

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  • Protein analysis method based on hydrophobic amino acid specific protease
  • Protein analysis method based on hydrophobic amino acid specific protease
  • Protein analysis method based on hydrophobic amino acid specific protease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Short-chain hydrophobic amino acid-specific protease for the analysis of myoglobin

[0017] (1) Take 0.2mg of Myoglobin and dissolve it in 200μL of 50mM Tris-HCl, 8M Urea, 150mMNaCl, 20mM CaCl 2 In the solution, the pH is 5.0, so that the concentration of Myoglobin in the solution is 1 mg / mL;

[0018] (2) Add the Myoglobin solution system in the above step (1) to a 30K ultrafiltration tube, and centrifuge at 10000g for 30min;

[0019] (3) Add 100 μL of 50 mM Tris-HCl, 150 mM NaCl, 20 mM CaCl to the ultrafiltration tube of the above step (2) 2 solution, the pH is 5.0, the Urea concentration is lower than 2M, the ratio of protein to enzyme is 20:1, add 10 μg of the protease, and incubate overnight at 20°C;

[0020] (4) Centrifuge the ultrafiltration tube with the Myoglobin solution after enzymolysis in the above step (3) at 10000 g for 30 min, and collect about 100 μL of proteolysis solution;

[0021] (5) Add 100 μL of 50 mM Tris-HCl, 150 mM NaCl, 20 mM CaCl to the ult...

Embodiment 2

[0025] Hydrolysis of bovine serum albumin (BSA) with short-chain hydrophobic amino acid specific protease and detection

[0026] (1) Take 0.2mg of BSA, dissolve in 200μL of 200mM Tris-HCl, 8M Urea, 200mMNaCl, 1mMCaCl 2 In the solution, the pH is 9.0, so that the BSA concentration in the solution is 1mg / mL;

[0027] (2) Add 500mM DTT 16μL to the BSA solution obtained in the above step (1) to make the final concentration of DTT 40mM, incubate at 100°C for 5min, then add 500mM IAA32μL, the final concentration is 80mM, and incubate at 25°C in the dark for 40min , to open the disulfide bond;

[0028] (3) Add the BSA solution system processed in the above step (2) into a 30K ultrafiltration tube, and centrifuge at 10000g for 30min;

[0029] (4) Add 100 μL of 200 mM Tris-HCl, 200 mM NaCl, 1 mM CaCl to the ultrafiltration tube of the above step (3) 2 solution, the pH is 9.0, the concentration of Urea is lower than 0.5M, the ratio of protein to enzyme is 50:1, add 4 μg of the protea...

Embodiment 3

[0035] The proteolytic enzyme Scanase digests the whole protein of HeLa cells and detects:

[0036] (1) HeLa cells were lysed to extract 0.2 mg of whole protein, with a volume of 200 μL, added to a 30K ultrafiltration tube, and centrifuged at 10,000 g for 30 min;

[0037] (2) Add 200μL of 50mM Tris-HCl, 8M Urea, 150mMNaCl, 20mM CaCl to the ultrafiltration tube of the above step (1) 2 Solution, pH 8.0, centrifuged at 10000g for 30min;

[0038] (3) repeat above-mentioned step (2) twice again;

[0039] (4) Add 200 μL of 50 mM Tris-HCl, 150 mM NaCl, 20 mM CaCl to the ultrafiltration tube of the above step (3) 2 Solution, pH 8.0, centrifuged at 10000g for 30min;

[0040] (5) repeat above-mentioned step (4) twice again;

[0041] (6) Add 100 μL of 50 mM Tris-HCl, 150 mM NaCl, 20 mM CaCl to the ultrafiltration tube of the above step (5) 2 solution, pH 8.0;

[0042] (7) Add 8 μL of 500 mM DTT to the whole protein solution of HeLa cells obtained in the above step (6) to make the f...

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Abstract

The invention provides a protein analysis method based on hydrophobic amino acid specific protease. The method firstly adopts short-chain hydrophobic amino acid specific protease for efficient enzymatic hydrolysis on the hydrophobic structural region of a protein; and then, a liquid chromatography-mass spectrography analysis technique is adopted for high throughput sequencing on peptides producedby enzyme digestion. Enzyme digestion sites for the adopted short-chain hydrophobic amino acid specific protease are mainly alanine, valine, leucine, isoleucine and other short-chain hydrophobic aminoacids, and the enzyme digestion specificity for the above four amino acids is up to 85%. The identification quantity of the proteins in a responsible biological sample and the coverage rate of the protein sequence analysis are remarkably improved, accurate analysis of post-translation modification information of the protein is facilitated, and the accuracy and the application range of the proteomic analysis technology are improved.

Description

technical field [0001] The invention belongs to the field of protein analysis, in particular to a protein analysis method based on hydrophobic amino acid specific protease. Background technique [0002] Protein is the main bearer of life activities, and its type, abundance and post-translational modification changes will directly affect cell function. Current proteomics analysis techniques based on liquid chromatography-mass spectrometry usually need to degrade proteins into peptides that are easy to analyze. Proteases are a class of enzymes that can hydrolyze proteins into polypeptides. Proteases are highly selective in the selection of substrates, and a protease can only hydrolyze peptide bonds containing one or more specific amino acids in a protein sequence. For example, trypsin, which is most widely used in the field of protein analysis, can only specifically cleave the C-terminus of the peptide bond containing arginine (Arginine, R) or lysine (Lysine, K) residues. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N33/68
Inventor 王方军孙彬文刘哲益
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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