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Enzymatic hydrolysis of proteins and glycoproteins

A protein and glycoprotein technology, applied in the fields of biology and chemistry, can solve the problems of loss of protein and enzyme, time-consuming, complex enzymatic hydrolysis process, etc., and achieve the effect of fast enzymatic hydrolysis and easy operation.

Active Publication Date: 2022-07-12
张怀远
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) The traditional enzymatic hydrolysis method: the enzymatic hydrolysis time is 12 hours or more, the disadvantage of this method: time-consuming, and generally rarely used for the enzymatic hydrolysis of small volume or small quality proteins
Disadvantages of this method: consume solid materials and enzymes, complicate the simple enzymatic hydrolysis process, introduce materials that are not conducive to subsequent scientific research, and lose proteins and enzymes (solid material surface adsorption), and are not conducive to small volume or small Enzymatic hydrolysis of high quality protein
Disadvantages of these methods: additional equipment is required, the enzymatic hydrolysis operation is complicated, and it is not conducive to field work

Method used

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  • Enzymatic hydrolysis of proteins and glycoproteins
  • Enzymatic hydrolysis of proteins and glycoproteins
  • Enzymatic hydrolysis of proteins and glycoproteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. In situ rapid proteolysis of the target plate

[0073] 1. Enzymatic hydrolysis of BSA

[0074] The BSA used is the product of Sigma-Aldrich Company.

[0075] a) NH 4 HCO 3 Concentration optimization

[0076] 1. Weigh 100mg of BSA standard protein with 100mL ddH 2 Dissolve O, put it into boiling water and boil for 10 minutes, then put it into an ice-water mixture to cool to obtain a 1 mg / mL denatured BSA solution; use deionized water to dissolve Tripsin to obtain a Tripsin aqueous solution with a Tripsin concentration of 100 ng / μL;

[0077] 2. Dilute 1 mg / mL denatured BSA solution with deionized water to obtain a denatured BSA solution with a BSA concentration of 100 ng / μL; 1 μL 100 ng / μL denatured BSA solution, 1 μL 100 ng / μL Tripsin aqueous solution, 1 μL NH 4 HCO 3 50mM NH 4 HCO 3 The aqueous solution and 7 μL of deionized water were quickly mixed to obtain a reaction mixture. In the reaction mixture, the concentrations of BSA and Tripsin were both ...

Embodiment 2

[0099] Example 2. Rapid enzymolysis of protein in liquid

[0100] 1. Weigh 200mg of BSA standard protein with 100mL ddH 2 O was dissolved, boiled in boiling water for 10 minutes, and then cooled in an ice-water mixture to obtain a 2 mg / mL denatured BSA solution;

[0101] 2. Dilute the 2 mg / mL denatured BSA solution in step 1 with deionized water to obtain a 400 ng / μL denatured BSA solution; take 25 μL of the 400 ng / μL denatured BSA solution, add 50 μL 50 mM NH to it 4 HCO 3 Aqueous solution and 25μL 200ng / μL Tripsin aqueous solution, after mixing evenly (the mass ratio of BSA and Tripsin in the reaction system obtained after mixing is 2:1, the concentration of Tripsin is 50ng / μL, NH 4 HCO 3 The concentration of 25mM) was incubated for 5 minutes at 37°C and 1200rpm (to increase the contact opportunity of protein and enzyme, using a dry thermostat (THERMO-SHAKER)) to obtain the enzymatic hydrolysis product.

[0102] 3. After the enzymatic hydrolysis in step 2, dilute the enz...

Embodiment 3

[0105] Embodiment 3, the comparison of the enzymatic hydrolysis method of the present invention and the traditional enzymatic hydrolysis method

[0106] 1. Traditional methods

[0107] 1. Weigh 200mg of BSA standard protein with 100mL ddH 2 O was dissolved, boiled in boiling water for 10 minutes, and then cooled in an ice-water mixture to obtain a 2 mg / mL denatured BSA solution.

[0108] 2. Dilute the 2 mg / mL denatured BSA solution in step 1 with deionized water to obtain a 20 ng / μL denatured BSA solution; take 25 μL of the 20 ng / μL denatured BSA solution, add 50 μL of 50 mM NH to it 4 HCO 3 Aqueous solution and 25 μL 0.4ng / μL Tripsin aqueous solution, after mixing evenly (the mass ratio of BSA and Tripsin in the reaction system obtained after mixing is 50:1, NH 4 HCO 3 The concentration of 25mM) was incubated at 37°C and 1200rpm for 12 hours to obtain the enzymatic hydrolysis product.

[0109] 3. After the enzymatic hydrolysis in step 2, take 1 μL of the enzymatic hydrol...

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Abstract

The invention discloses an enzymatic hydrolysis method of protein and glycoprotein. The enzymatic hydrolysis method of protein or glycoprotein disclosed in the invention comprises: incubating the protein or glycoprotein and protease in a reaction system for 0.5-15 minutes to complete the enzymatic hydrolysis of the protein or glycoprotein; the reaction system contains NH 4 HCO 3 , NH in the reaction system 4 HCO 3 The concentration of the protein is greater than 0mM and less than or equal to 100mM; the mass ratio of protein or glycoprotein to protease in the reaction system is 1:(0.0001-3); the incubation time is 0.5-10 minutes, and the temperature is 4-80°C. The method of the present invention can be applied to fields that require protein enzymatic hydrolysis: such as identification of proteins, obtaining specific glycopeptides, identifying peptide segments with mutated amino acids, and the like.

Description

technical field [0001] The present invention relates to a method for enzymatic hydrolysis of proteins and glycoproteins in the fields of biology and chemistry. Background technique [0002] The significance of studying protein enzymatic hydrolysis is as follows: 1. Obtain protein peptides by enzymatic cleavage of proteins, and obtain peptide fingerprints by mass spectrometry to identify proteins, which can produce protein identification kits or protein enzymatic hydrolysis kits; 2. Obtain Conduct scientific research on specific enzymatic peptides, such as obtaining glycopeptides to be studied, obtaining peptides with amino acid mutations, etc.; 3. Determine the post-translational modification sites of the research protein, such as: glycosylation sites, methyl groups sites, acetylation sites, etc. [0003] The current methods of protein enzymatic hydrolysis include: [0004] (1) Traditional enzymatic hydrolysis method: The enzymatic hydrolysis time is 12 hours or more. The ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/06
CPCC12P21/06
Inventor 张怀远丁玮云欧凌
Owner 张怀远
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