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

A peptide fragment and kit technology, which is applied in the preparation methods of peptides, the preparation of test samples, biochemical equipment and methods, etc. Analysis accuracy, effect of simplified condition setting

Active Publication Date: 2016-04-20
SHIMADZU CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ELISA method is a method for the detection of a single protein (antigen or antibody), and cannot detect multiple different proteins at the same time.
In order to detect multiple proteins by ELISA, it is necessary to prepare specific antibodies for each detection object and set the concentration measurement conditions, which requires a lot of effort and cost.
In addition, since the ELISA method utilizes an antigen-antibody reaction, the following problems are known: the measurement result may be affected by the combination drug and cross-reaction with metabolites, and it is difficult to store some antibody drugs for the specimen, etc.
However, there are problems that the more complicated the sample becomes, the more various separation modes need to be combined, the more high-precision experimental equipment is required, or the setting of analysis conditions takes a lot of time.
However, since the method of the above-mentioned Patent Document 1 requires the Fab domain or F(ab') 2 Purification of the domain is followed by protease treatment, so it takes a lot of time and cost to prepare a sample for mass spectrometry, and it is difficult to call it an easy method

Method used

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

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Experimental program
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preparation example Construction

[0055] In the method for producing a peptide fragment of the present invention, a matrix protein to be cleaved is immobilized in pores of a porous body, and the porous body immobilized with the matrix protein is brought into contact with microparticles having protease immobilized on the surface in a liquid. figure 1 It is a conceptual diagram for explaining the principle of protease digestion in this invention.

[0056] In particles 10 (average particle size D 1 ) with protease 15 immobilized on the surface. The porous body 20 has a plurality of pores 29 (average pore diameter D 2 ), and matrix protein 25 is immobilized in the pores. Thus, in the method of the present invention, both the protease 15 and the matrix protein 25 are immobilized on the solid phase as minute domains, and protease digestion is performed by contacting the solid phases with each other.

[0057] Average particle diameter D of microparticles 10 1 greater than the average pore diameter D of the porou...

Embodiment

[0121] The following shows an experimental example in which human immunoglobulin G (IgG) and trastuzumab (trastuzumab (trade name: Herceptin)) were digested with protease according to the method of the present invention, and the resulting peptide fragment samples were subjected to mass spectrometry. It should be noted that the present invention is not limited to the following examples.

[0122] Hereinafter, description of % means % by weight unless otherwise specified. Reagents and the like used in this experimental example are as follows.

[0123] Trypsin (sequence grade, promega)

[0124] Lysyl endopeptidase (mass spec grade, Wako Pure Chemical Industries)

[0125] 2-Morpholineethanesulfonic acid, (MES, Doujin Chemical)

[0126] 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, Doujin Chemical)

[0127] Tris(hydroxymethyl)aminomethane (Tris, Wako Pure Chemical Industries)

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Abstract

A method for preparing peptide fragments by cleaving a protein using a protease. The method according to the present invention comprises a step for contacting, in a liquid, a porous body (20), in which the substrate protein (25) to be cleaved is immobilized in pores (29), with microparticles (10) which carry the protease (15) immobilized on the surface thereof. In the present invention, the average particle diameter of the microparticles (10) is larger than the average pore diameter of the porous body (20). According to the method of the present invention, the substrate protein (25) that is an antibody can be site-specifically cleaved. By analyzing the peptide fragments that are obtained by the above method by mass spectrometry, etc., the antibody protein can be detected or quantitatively analyzed.

Description

technical field [0001] The present invention relates to a method for preparing peptide fragments by site-selectively cleaving proteins such as antibodies with a protease, and a kit for preparing peptide fragments used in the method. Furthermore, the present invention relates to an analysis method for detecting and quantifying a protein by analyzing a peptide fragment prepared by the method by mass spectrometry or the like. Background technique [0002] Demand for antibody drugs is rapidly expanding, and the importance of easily quantifying the concentration of antibodies in blood is increasing in clinical settings. Although the accurate measurement of blood drug concentration after administration of antibody drugs has not been paid attention to until now, the relationship between blood drug concentration and survival time of trastuzumab (trastuzumab (trade name: Herceptin)) has been found in clinical trials with the expansion of gastric cancer application. After there is a ...

Claims

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Application Information

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IPC IPC(8): G01N27/62G01N1/28
CPCC07K1/107C07K1/12C07K17/00C12P21/06G01N2333/948C12N11/00G01N1/405G01N33/6851G01N33/6857G01N2333/976G01N2560/00
Inventor 岛田崇史深尾典子青木智景佐藤孝明
Owner SHIMADZU CORP
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