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

Patent metadata

Field	Value
Publication number	US-2016252522-A1
Application number	US-201615063774-A
Country	US
Kind code	A1
Filing date	Mar 8, 2016
Priority date	Sep 9, 2013
Publication date	Sep 1, 2016
Grant date	—

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 6 , wherein the immobilizing is performed such that the protein having the linker molecule bound thereto has a molecular size which is 0.5 times to 1.5 times greater than the average pore diameter of the pores.

8 . 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.

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

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

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

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

13 . The method of claim 9 , 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.

14 . The method of claim 11 , 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.

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

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

17 . 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.

18 . The method of claim 11 , 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.

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

20 . The method of claim 18 , wherein the average particle diameter of the particles is 170 nm or greater.

21 . The method of claim 18 , wherein the average particle diameter of the particles is in a range of from 120 nm to 1 μm.

22 . The method of claim 20 , wherein the average particle diameter of the particles is in a range of from 170 nm to 300 nm.

23 . A kit for proteolyzing a protein, comprising:
at least one porous body having a plurality of pores in which a protein is to be immobilized; and a plurality of particles having an average particle diameter larger than an average pore diameter of the pores, wherein the particles have a surface on which a protease has been immobilized or to be immobilized.

24 . The kit of claim 23 , wherein the particles have a protease immobilized on the surface of the particles.

25 . The kit of claim 23 , wherein the average particle diameter of the particles is 100 nm or greater and the average pore diameter of the pores in the porous body is in a range of from 30 nm to 150 nm, provided that the average particle diameter is larger than the average pore diameter.

26 . A method of quantitating a protein in a sample, comprising:
proteolyzing a protein in a sample by the method of claim 1 such that a plurality of peptide fragments including a peptide fragment specific to the protein are produced from the protein; and subjecting the peptide fragments to a mass spectrometric analysis to detect the peptide fragment.

27 . The method of claim 26 , wherein the protein is an antibody, and the peptide fragments include at least a portion of an amino acid sequence of a complementarity determining region of the antibody.

Assignees

• Shimadzu Corp

Inventors

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

Classifications

• C07K1/12

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

• C07K1/107 Primary

  by chemical modification of precursor peptides · CPC title

• G01N1/405

  by adsorption or absorption · CPC title

• C12N11/00

  Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof · CPC title

• G01N33/6857 Primary

  Antibody fragments · CPC title