Method for preparing peptide fragments, kit for preparing peptide fragments to be used therein, and analysis method

Patent metadata

Field	Value
Publication number	US-11022616-B2
Application number	US-201916683560-A
Country	US
Kind code	B2
Filing date	Nov 14, 2019
Priority date	Sep 9, 2013
Publication date	Jun 1, 2021
Grant date	Jun 1, 2021

Abstract

A method of proteolyzing a protein, including immobilizing a protein in at least one pore of a porous body, and contacting the protein immobilized in the pore and a protease immobilized on a solid surface such that the protease selectively accesses a site of the protein and proteolyzes the protein at the site.

First claim

What is claimed is:

1. A method of proteolyzing a protein, comprising:
immobilizing a protein in at least one pore of a porous body; and
contacting the protein immobilized in the pore and a protease immobilized on a solid surface such that the protease selectively accesses a site of the protein and proteolyzes the protein at the site.

2. The method of claim 1 , wherein the solid surface is a surface of at least one particle.

3. The method of claim 2 , wherein the at least one pore is a plurality of pores, the at least one particle is a plurality of particles, and the plurality of particles has an average particle diameter larger than an average pore diameter of the plurality of pores.

4. The method of claim 3 , wherein the contacting is performed in a liquid.

5. The method of claim 1 , wherein the immobilizing comprises immobilizing the protein at a site different from the site of the protein at which the protein is proteolyzed.

6. The method of claim 1 , wherein the immobilizing comprises immobilizing the protein in the pore through a linker molecule that site-specifically interacts with the protein.

7. The method of claim 2 , wherein the protease is immobilized on the surface of the particle through a spacer molecule that binds with the protease.

8. The method of claim 1 , wherein the protein is an antibody.

9. The method of claim 8 , wherein the antibody is a monoclonal antibody.

10. The method of claim 4 , wherein the protein is an antibody.

11. The method of claim 10 , wherein the antibody is a monoclonal antibody.

12. The method of claim 8 , wherein the immobilizing comprises immobilizing an Fc domain of the antibody, and the contacting is performed such that a Fab domain of the antibody is site-selectively proteolyzed by the protease.

13. The method of claim 10 , wherein the immobilizing comprises immobilizing an Fc domain of the antibody, and contacting is performed such that a Fab domain of the antibody is site-selectively proteolyzed by the protease.

14. The method of claim 1 , wherein the protease is trypsin.

15. The method of claim 4 , wherein the protease is trypsin.

16. The method of claim 4 , wherein the average particle diameter of the particles is 100 nm or greater and the average pore diameter of the pores is in a range of from 30 nm to 150 nm, provided that the average particle diameter is larger than the average pore diameter.

17. The method of claim 10 , wherein the average particle diameter of the particles is 120 nm or greater, and the average pore diameter of the pores is in a range of from 50 nm to 100 nm.

18. The method of claim 17 , wherein the average particle diameter of the particles is 150 nm or greater.

19. The method of claim 17 , wherein the average particle diameter of the particles is 170 nm or greater.

20. The method of claim 17 , wherein the average particle diameter of the particles is in a range of from 120 nm to 1 μm.

21. The method of claim 19 , wherein the average particle diameter of the particles is in a range of from 170 nm to 300 nm.

Assignees

• Shimadzu Corp

Inventors

• Shimada Takashi
• Iwamoto Noriko
• Aoki Chikage
• Sato Taka-Aki

Classifications

• C07K1/107 Primary

  by chemical modification of precursor peptides · CPC title

• C12P21/06

  produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00) · CPC title

• G01N33/6857 Primary

  Antibody fragments · CPC title

• G01N33/6851

  Methods of protein analysis involving laser desorption ionisation mass spectrometry · CPC title

• G01N2333/948

  acting on peptide bonds (3.4) · CPC title