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Method for carrying out protein enzymatic hydrolysis

A protein and protein solution technology, applied in the field of protein enzymatic hydrolysis, can solve the problems affecting the speed of large-scale identification of proteins and the lack of stable means for preparation, and achieve the effect of fast speed, simple steps, and improved enzymatic activity

Active Publication Date: 2015-08-12
GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In actual proteomics experiments, no matter which analysis strategy is used, the step of protein enzymatic hydrolysis is included. At present, the efficiency controllable programming scheme is used for protein separation and identification, but for sample preparation such as protein enzymatic hydrolysis The lack of stable means of work seriously affects the speed of large-scale identification of proteins

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  • Method for carrying out protein enzymatic hydrolysis
  • Method for carrying out protein enzymatic hydrolysis

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Embodiment 1

[0033] A method of enzymatically hydrolyzing protein, adding water to the protein and stirring it evenly, putting it into an electrophoresis tank, electrophoresis at 80V, collecting the protein solution after electrophoresis, dissolving the protein with an enzymolysis buffer solution containing trifluoroethanol, and then reducing the protein and alkylation treatment, adjusting the pH of the treated protein solution to 7.5, then adding trypsin accounting for 90% of the volume of the entire solution, and enzymatically hydrolyzing it to obtain a polypeptide product;

[0034] Wherein, the working electrode in the electrophoresis tank is an electrode modified by redox enzyme and zinc-aluminum layered double hydroxide.

[0035] Wherein, the enzymolysis buffer solution is composed of ammonium bicarbonate, trifluoroethanol and acetonitrile in a mass ratio of 2:40:8.

[0036] Wherein, the reagent for adjusting the pH value is ammonium bicarbonate solution with a mass fraction of 2%.

...

Embodiment 2

[0052] A method of enzymatically hydrolyzing protein, adding water to the protein and stirring it evenly, putting it into an electrophoresis tank, electrophoresis at 120V, collecting the protein solution after electrophoresis, dissolving the protein with an enzymolysis buffer solution containing trifluoroethanol, and then reducing the protein and alkylation treatment, adjusting the pH of the treated protein solution to 8.5, then adding trypsin accounting for 98% of the volume of the entire solution, and enzymatically hydrolyzing it to obtain a polypeptide product;

[0053] Wherein, the working electrode in the electrophoresis tank is an electrode modified by redox enzyme and zinc-aluminum layered double hydroxide.

[0054] Wherein, the enzymolysis buffer solution is composed of ammonium bicarbonate, trifluoroethanol and acetonitrile in a mass ratio of 2:60:15.

[0055] Wherein, the reagent for adjusting the pH value is ammonium bicarbonate solution with a mass fraction of 4%. ...

Embodiment 3

[0071] A method of enzymatically hydrolyzing protein, adding water to the protein and stirring it evenly, putting it into an electrophoresis tank, electrophoresis at a voltage of 100V, collecting the protein solution after electrophoresis, dissolving the protein with an enzymolysis buffer solution containing trifluoroethanol, and then reducing the protein and alkylation treatment, adjusting the pH of the treated protein solution to 8, then adding trypsin accounting for 96% of the volume of the entire solution, and enzymatically hydrolyzing it to obtain a polypeptide product;

[0072] Wherein, the working electrode in the electrophoresis tank is an electrode modified by redox enzyme and zinc-aluminum layered double hydroxide.

[0073] Wherein, the enzymolysis buffer solution is composed of ammonium bicarbonate, trifluoroethanol and acetonitrile in a mass ratio of 2 ∶ 50 ∶ 10 compositions.

[0074] Wherein, the reagent for adjusting the pH value is ammonium bicarbonate solutio...

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Abstract

The invention discloses a method for carrying out enzymatic hydrolysis. The method comprises the following steps: adding water into protein, evenly stirring, transferring the mixture to an electrophoresis tank, carrying out electrophoresis under a voltage of 80 to 120 V, collecting the protein solution after electrophoresis, dissolving the protein by an enzymatic hydrolysis buffer solution containing trifluoroethanol, subjecting the protein to reduction and alkylation treatments, adjusting the pH value of processed protein to 7.5-8.5, then adding trypsase accounting for 90 to 98% of the protein solution, and carrying out enzymatic hydrolysis to obtain polypeptide; wherein the work electrode in the electrophoresis tank is modified by oxidation-reduction enzyme and Zn-Al layered double hydroxides. The provided method can obtain peptides suitable for liquid chromatogram-mass spectrum (LS-MS) analysis, and provides stable and reliable samples for proteomics analysis.

Description

technical field [0001] The invention relates to the field of protein enzymolysis, more specifically, the invention relates to a method for stably and rapidly enzymatically hydrolyzing proteins. Background technique [0002] Proteins are the key structural and functional molecules in organisms, the executors of life activities and the embodiment of life phenomena. Proteome refers to all proteins expressed by genes in a cell or a tissue under specific conditions. The analysis and identification of these proteins is called proteomics. [0003] Proteins are the executors of gene functions, and their expression patterns and functional analysis have become one of the core contents of research in the post-genome era. The focus of proteome research is on all proteins in a cell line, an organ or an organism, and is committed to elucidating the information and interaction relationships of all protein types. Through proteomics research, high-sensitivity biomarkers can be found, new p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 潘丽梅韦荣昌付强马小军白隆华黄芩芬
Owner GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS
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