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

Patent metadata

Field	Value
Publication number	US-11650210-B2
Application number	US-202117231141-A
Country	US
Kind code	B2
Filing date	Apr 15, 2021
Priority date	Sep 9, 2013
Publication date	May 16, 2023
Grant date	May 16, 2023

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. An analytical method, comprising:
proteolyzing a protein in a sample such that a peptide fragment is produced; and
identifying or quantifying the peptide fragment in the sample by mass spectrometry,
wherein the proteolyzing includes immobilizing the 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 analytical method of claim 1 , wherein the mass spectrometry is one of a quadrupole method, an ion trap method, and a time-of-flight method.

3. The analytical method of claim 1 , wherein the mass spectrometry is one that conducts MS/MS analysis, or MS 3 or higher-order MS.

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

5. The analytical method of claim 4 , 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.

6. The analytical method of claim 5 , wherein the contacting is performed in a liquid.

7. The analytical 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.

8. The analytical 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.

9. The analytical method of claim 4 , wherein the protease is immobilized on the surface of the particle through a spacer molecule that binds with the protease.

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

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

12. The analytical method of claim 6 , wherein the protein is an antibody.

13. The analytical method of claim 12 , wherein the antibody is a monoclonal antibody.

14. The analytical method of claim 10 , 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.

15. The analytical method of claim 12 , 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.

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

17. The analytical method of claim 6 , wherein the protease is trypsin.

18. The analytical method of claim 6 , 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.

19. The analytical method of claim 12 , 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.

20. The analytical method of claim 19 , wherein the average particle diameter of the particles is 150 nm or greater.

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

22. The analytical method of claim 19 , wherein the average particle diameter of the particles is in a range of from 120 nm to 1 μm.

23. The analytical method of claim 21 , 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

• G01N2333/948

  acting on peptide bonds (3.4) · CPC title

• C07K1/107 Primary

  by chemical modification of precursor peptides · CPC title

• C07K1/12

  by hydrolysis {, i.e. solvolysis in general} · CPC title

• G01N2560/00

  Chemical aspects of mass spectrometric analysis of biological material · CPC title

• G01N33/6857 Primary

  Antibody fragments · CPC title