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

Patent metadata

Field	Value
Publication number	US-10539571-B2
Application number	US-201615063774-A
Country	US
Kind code	B2
Filing date	Mar 8, 2016
Priority date	Sep 9, 2013
Publication date	Jan 21, 2020
Grant date	Jan 21, 2020

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 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 modified by a spacer such that a protease has been immobilized or is to be immobilized on the surface by specific binding with the spacer,
a ligand molecule is immobilized in the pores of the porous body,
the ligand molecule is capable of site-specifically interacting with and controlling an orientation of the protein to be immobilized, and
the average particle diameter or the average pore diameter is sized such that each of the pores has a limit to the region accessible to the protease, and that the protein is to be immobilized with a tip portion extending into the region accessible to the protease.

2. The kit of claim 1 , wherein the particles have a protease immobilized on the surface of the particles modified by the spacer.

3. The kit of claim 1 , 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.

4. The kit of claim 1 , wherein the ligand molecule is capable of site-specifically interacting with Fc domain of an antibody.

5. The kit of claim 1 , wherein the ligand molecule is at least one selected from the group consisting of protein A and protein G.

6. The kit of claim 1 , wherein the spacer allows specific binding with the protease without deactivating the protease.

7. The kit of claim 1 , wherein the spacer has a molecular diameter of 5 nm or less.

8. The kit of claim 1 , wherein the spacer has a molecular diameter of 2 nm or less.

9. The kit of claim 1 , wherein the spacer has a molecular weight of 2000 or less.

10. The kit of claim 2 , wherein the spacer has a molecular weight of 800 or less.

11. The kit of claim 1 , wherein the spacer has one of an amino group, an amide group, an ester group, an epoxy group, a carboxyl group, biotin, avidin, and a chelate.

12. The kit of claim 1 , wherein the spacer has an ester group.

13. The kit of claim 1 , wherein the spacer has an activated ester group.

14. A kit for proteolyzing a protein, comprising:
at least one porous body having a plurality of pores in which a protein is 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,
a ligand molecule is immobilized in the pores of the porous body,
the ligand molecule immobilized in the pores is capable of site-specifically interacting with the immobilized protein, thereby an orientation of the protein immobilized in the pores is controlled,
the average particle diameter or the average pore diameter is sized such that the protease immobilized on the surface of the particles has access to the protein immobilized in the pores of the porous body,
the pores each have a limit to the region accessible to the protease, and
the protein immobilized in the pores has a tip portion extending into the region accessible to the protease.

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

• G01N2333/976

  Trypsin; Chymotrypsin · CPC title

• G01N2560/00

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

• G01N2333/948

  acting on peptide bonds (3.4) · CPC title