JP6152908B2 - Method for preparing and analyzing peptide fragment - Google Patents

Description

  The present invention relates to a method for preparing peptide fragments by regioselectively cleaving a protein such as an antibody using a protease. Furthermore, the present invention relates to an analysis method for detecting and quantifying proteins by analyzing peptide fragments prepared by the method by mass spectrometry or the like.

  The demand for antibody drugs is rapidly expanding, and the importance of simply quantifying the antibody concentration in blood in clinical settings is increasing. Previously, it was not important to accurately measure the blood concentration after administration of the antibody drug, but in clinical trials associated with the expansion of gastric cancer application of trastuzumab (trade name: Herceptin), its blood concentration and survival Since a significant difference was found in the period, it has become essential to measure the blood concentration of an antibody in clinical trials of antibody drugs. For example, identification of antibody drugs and quantification of their blood concentrations are also required in quality control such as pharmacokinetic confirmation and identity confirmation with the original drug in clinical trials of generic drugs. .

  ELISA (enzyme-linked immunosorbent assay) using antigen-antibody reaction is widely used for detection and quantification of antigens and antibodies in blood in clinical trials and the like because of its excellent specificity and quantification. . However, the ELISA method is intended to detect a single protein (antigen or antibody), and cannot detect many different proteins simultaneously. In order to detect a large number of proteins by the ELISA method, it is necessary to prepare specific antibodies for each detection target and set the concentration measurement conditions, which requires enormous labor and cost. In addition, since the ELISA method uses an antigen-antibody reaction, cross-reactions with concomitant drugs and metabolites can affect the measurement results, and some antibody drugs are difficult to apply to stored samples. The problem is known.

  Therefore, it is desired to develop an analytical technique that can be applied to various antibodies for general purposes. With the development of proteomics, technologies capable of comprehensively detecting and quantifying proteins by mass spectrometry have been developed. In recent years, it has become possible to analyze macrobiomolecules such as antibodies. In mass spectrometry, it is necessary to ionize a molecule to be measured, and it is often difficult to analyze a giant biomolecule such as an antibody as it is by mass spectrometry. Therefore, a protein is cleaved (fragmented) by protease digestion, and a peptide fragment having an amino acid sequence specific to the protein to be analyzed is selected from the fragmented peptides, and detected and quantified by a mass spectrometer. The method is adopted. In general, in order to improve the digestion efficiency by protease, protease digestion is performed after denaturing the substrate protein in a high-concentration urea or guanidine solution.

  When mass spectrometry is performed by fragmenting a protein such as an antibody by protease digestion, it is important to selectively detect a peptide fragment to be detected. However, it may be difficult to detect a specific peptide fragment derived from the protein to be analyzed from a biological sample containing a wide variety of contaminants. That is, since biological samples such as blood contain a wide variety of contaminants, when a protease is added to the biological sample, the protein derived from the contaminants is also digested with the protease and a huge number of peptide fragments are generated. In order to selectively detect and quantify the target peptide fragment derived from the detection target (for example, a specific antibody), operations such as separation and concentration of the peptide fragment are required before being subjected to mass spectrometry. Moreover, if a peptide fragment having an amino acid sequence common to the peptide fragment of the detection target protein is produced from a protein other than the detection target, it may cause erroneous detection or a decrease in quantitativeness. Therefore, with an increase in the number of peptide fragments generated from a biological sample, selection of peptide fragments to be detected tends to be more difficult.

  In view of the complexity of such biological samples, the sample is purified by liquid chromatography (LC) and then subjected to mass spectrometry, multistage mass spectrometry that causes ion cleavage by collision-induced dissociation (CID), etc. Alternatively, a method for quantifying a specific protein in a complex sample such as a biological sample has been developed by combining these (multiple reaction monitoring, MRM). However, as the sample becomes more complicated, it becomes necessary to combine various separation modes, and there is a problem that a highly accurate experimental apparatus is required and it takes much time to set analysis conditions.

In addition, since a large number of peptide fragments are produced by protease digestion even when a purified sample is used for a large protein such as an antibody, a peptide fragment having an amino acid sequence peculiar to the detection target is selectively selected from them. It tends to be difficult to detect and quantify. In view of such a problem, a method has been developed that reduces the number (type) of peptide fragments in a sample by regioselectively digesting a protein to be analyzed with a protease, thereby improving analysis accuracy and simplifying analysis. ing. For example, in Patent Document 1, an F (ab ′) ₂ fragment is produced by pepsin digestion of an antibody, and then the F (ab ′) ₂ fragment is further digested with a protease such as trypsin to obtain the complementarity determining region (CDR) of the antibody. ) Has been proposed, and peptide fragments containing CDRs are detected and quantified by mass spectrometry.

  Here, the structure of the antibody will be described. All antibodies have two heavy chains (H chains) and two light chains (L chains). One light chain and one heavy chain are connected by a disulfide bond to form a heterodimer, and two heterodimers are further connected by two disulfide (SS) bonds to form a “Y” -shaped heterotetramer. (See FIG. 2). The antibody has one Fc (Fragment, crystallizable) domain consisting of a heavy chain and two Fab (Fragment, antigen binding) domains consisting of a heavy chain and a light chain. The Fc domain and the Fab domain have a hinge part. Connected through.

  The Fc domain of an antibody mainly has a function (effector function) for inducing a reaction after the antibody binds to an antigen, and most antibodies derived from the same species share the same amino acid sequence of the Fc domain. . On the other hand, the Fab domain has a function in which the tip portion (N-terminal side) binds to an antigen. In the Fab domain, various changes in the amino acid sequence are observed so that the N-terminal portion can bind to various antigens. This region is called a variable region (V region), the light chain variable region is called a VL region, and the heavy chain variable region is called a VH region. The Fab domain and Fc domain other than the V region are regions with little change in amino acid sequence, and are referred to as constant regions (C regions). The constant region of the light chain is called the CL region, and the constant region of the heavy chain is called the CH region. The CH region is further divided into three regions, a CH1 region, a CH2 region, and a CH3 region. The heavy chain Fab domain consists of a VH region and a CH1 region, and the heavy chain Fc domain consists of CH2 and CH3. The hinge part is located between CH1 and CH2.

  The specificity of an antibody (ie, specific binding to an antigen) is determined by a combination of amino acid sequences in the V region. Each of the light and heavy chains has three complementarity determining regions (CDRs) within the V region of the Fab domain. CDRs are also referred to as hypervariable regions, and the amino acid sequence varies depending on the type of antibody. Since an antibody has three CDRs in each of a light chain and a heavy chain (a total of six kinds of CDRs), diversity that allows binding to various antigens occurs. In other words, the CDR is a region characterizing the antibody, and the antibody can be identified by specifying the amino acid sequence of the CDR of the antibody.

As described above, the Fab domain and Fc domain of an antibody are connected via a hinge part. Papain, a kind of protease, cleaves this hinge part, so that two Fab domains and one Fc domain are produced by papain digestion of the antibody. In addition, pepsin, which is a kind of protease, cleaves the Fc domain side (C-terminal side) of two disulfide bonds in the hinge part, so that F (ab ′) ₂ domain in which two Fab domains are bound by pepsin digestion A number of Fc domain fragments are produced.

In the method of Patent Document 1, a Fab domain or F (ab ′) ₂ domain is produced by pepsin digestion or papain digestion, and the Fab domain or F (ab ′) ₂ domain is regioselectively digested with protease. According to this method, the number of peptides produced by protease digestion can be reduced and peptides containing CDRs can be produced efficiently, so that detection and quantification of antibodies by mass spectrometry is simplified. Moreover, in patent document 2, it is reported that the digestion efficiency by these protease improves by using pepsin or papain and a specific ion together.

For the purpose of purifying the antibody Fab domain, a purification kit optimized for protease is commercially available. However, since the method of Patent Document 1 requires purification of the Fab domain or F (ab ′) ₂ domain and further protease treatment, it requires a lot of time and cost to prepare a sample for mass spectrometry. However, it is difficult to say that this is a simple technique. In addition, since digestion efficiency (fragmentation) differs depending on the type of substrate protein to be digested, the digestion efficiency is improved to simplify the preparation of peptide fragment samples for mass spectrometry. Is important.

  In recent years, as a method for improving the efficiency of protease digestion, a method of performing protease digestion in a microenvironment such as nanoparticles has attracted attention. For example, Non-Patent Document 1 reports an example in which trypsin is immobilized in the pores of a nylon porous membrane and albumin solution is passed through the porous membrane, thereby increasing the efficiency of albumin trypsin digestion. Yes. Non-Patent Document 2 reports that a protein having a small molecular weight can be selectively digested with trypsin by using mesoporous silica in which trypsin is fixed in the pores. In any of these methods, the protease is immobilized in the pores of the porous body, and the protease on the solid phase surface reacts with the substrate protein in the liquid phase. In this way, the efficiency of digestion is improved by immobilizing the protease to the solid phase in a microenvironment, because the protein tends to denature in a local and microenvironment such as the interface between the solid phase and the liquid phase. Further, it is considered that the contact probability between the substrate protein and the protease increases due to the perturbation of the solubility, the fluctuation width of the three-dimensional structure, etc., and the reaction probability is improved.

International Publication Pamphlet of WO2008 / 079914 JP 2011-130749 A

Fei Xu et.al., Anal. Chem., 2010, 82, 10045-10051. Qianhao Min et.al., Chem. Commun., 2010, 46 (33), 6144-6146.

  The method of Non-Patent Document 2 can selectively digest a protein having a small molecular weight with protease, but cannot be applied to selective digestion of a large protein such as an antibody. In addition, a method for performing protease digestion in a site-selective manner using a microreactor as disclosed in Non-Patent Document 1 and Non-Patent Document 2 has not been developed.

  As described above, in order to easily detect and quantify a protein by mass spectrometry, the protein to be analyzed is cleaved in a position-selective manner to efficiently produce peptide fragments specific to the protein to be detected. It is required to reduce the production amount of other peptide fragments. For example, in order to detect and quantitatively analyze antibodies, it is necessary to regioselectively digest the Fab domain, particularly the V domain of the Fab domain, with protease, thereby suppressing protease digestion of the Fc domain.

  As a result of intensive studies by the present inventors, it has been found that by fixing both a substrate protein such as an antibody and a protease to a solid phase, a site-selective protease digestion of the substrate protein can be realized, and the present invention has been achieved.

  The present invention relates to a method for preparing a peptide fragment for digesting a protein with a protease. In the method of the present invention, the substrate protein to be cleaved immobilized in the pores of the porous body and the protease immobilized on the solid surface are brought into contact so that the protease accesses the cleavage site of the substrate protein. It has a step (digestion step). The porous body in which the substrate protein is immobilized in the pores is obtained by a step of immobilizing the substrate protein to be cleaved in the pores of the porous body (substrate immobilization step). In the present invention, fine particles or the like are used as the solid phase. The particle diameter of the fine particles is preferably larger than the average pore diameter of the porous body.

  When the particle size of the fine particles is larger than the average pore size of the porous body, the protease immobilized on the surface of the fine particles can access the vicinity of the surface layer of the pores of the porous body, but accesses the deep position of the pores. Can not. Thus, since the accessible region of the protease is physically (spatial) restricted, the protease selectively accesses a specific site of the substrate protein immobilized in the pores of the porous body. . This makes it possible to perform protease selective digestion (fragmentation) of the substrate protein.

  In the present invention, the predetermined region of the substrate protein is preferably immobilized on the porous body. In this form, the region fixed to the porous body is located deep in the pores, and the region other than the fixed region is located near the surface layer of the pores. If a region different from the selective cleavage site of the substrate protein, that is, a region preferably not digested with rotase, is immobilized on the porous body, the selective cleavage site of the substrate protein located near the surface layer of the pore is placed on the surface of the fine particle. The immobilized protease accesses and protease digestion takes place. Therefore, a desired site of the substrate protein can be digested with protease in a regioselective manner.

  A linker molecule that interacts with a substrate protein in a site-specific manner is preferably immobilized in the pores of the porous body. The substrate protein is preferably immobilized in the pores of the porous body via a linker molecule. Examples of the linker molecule when the substrate protein is an antibody include protein G and protein A. Since these linker molecules bind site-specifically to the Fc region of the antibody, the Fc region of the antibody is immobilized on the porous body, and the Fab region is located near the surface layer of the pore. According to this form, the Fab region of the antibody can be selectively digested with protease.

  When the linker molecule is immobilized in the pores of the porous body, the molecular size of the state in which the linker molecule and the substrate protein are bound is about 0.5 to 1.5 times the average pore diameter of the porous body It is preferable that By adjusting the molecular size in this manner, the access probability between the protease immobilized on the surface of the fine particle and the selective cleavage site of the substrate protein can be increased, and the position selectivity of protease digestion can be improved.

  The surface of the fine particles is preferably modified with a spacer molecule that can bind to a protease, and the protease is preferably immobilized on the surface of the fine particle via the spacer molecule. By immobilizing the protease via the spacer molecule, the detachment of the protease from the surface of the fine particles is suppressed, and the position selectivity of protease digestion is enhanced. In addition, by adjusting the molecular size of the spacer, the protease can be selectively accessed at a desired position of the substrate protein, thereby enhancing the position selectivity.

  In the present invention, the protease immobilized on the surface of the fine particles is preferably trypsin or a combined system of trypsin and another protease. When the substrate protein is an antibody, trypsin is used alone, or in the case of a combined system, the amount of trypsin in the total protease is preferably 90% or more. In particular, when the substrate protein is an antibody, trypsin tends to selectively digest the Fab domain with protease and suppress protease digestion of the Fc domain.

  The average pore diameter of the porous body is preferably about 30 to 150 nm, and the average particle diameter of the fine particles is preferably 100 nm or more. In particular, when the substrate protein is an antibody, the Fab region can be cleaved more reliably and selectively if the average pore diameter and the average particle diameter are in the above ranges.

  The peptide fragment preparation kit used in the above method comprises a porous body having pores capable of immobilizing proteins and fine particles capable of immobilizing protease on the surface. The microparticles may be provided with a protease immobilized on the surface. By contacting the porous body of this kit with a sample (for example, a specimen such as blood), the target substance (substrate protein such as an antibody) in the sample can be immobilized in the pores of the porous body. The substrate protein is regioselectively digested with the protease by contacting the porous body after the substrate protein is immobilized with the microparticles on which the protease is immobilized on the surface.

  By analyzing the peptide fragment obtained by the above method by mass spectrometry or the like, substrate protein can be detected (identified) or quantified. In the present invention, since the substrate protein is site-selectively protease-digested, the types of peptide fragments contained in the measurement sample can be greatly reduced. Therefore, the condition setting of mass spectrometry can be simplified, and improvement in analysis accuracy can be expected.

  For example, when the substrate protein is an antibody, the method of the present invention enables position-selective protease digestion of the Fab region including the complementary determining region, so that at least a partial sequence of the complementarity determining region of the antibody A peptide fragment containing can be detected. Since the complementarity determining region has an amino acid sequence specific to each antibody, the antibody can be detected and quantified by analyzing a peptide fragment containing the sequence of the complementary determining region.

  According to the present invention, a peptide fragment can be obtained by site-selective protease digestion of a protein such as an antibody by a simple method. When the method of the present invention is applied to an antibody, digestion of the Fc region of the antibody is suppressed, the Fab region containing CDR is selectively protease-digested, and a sample of a peptide fragment containing the CDR amino acid sequence important for antibody identification The concentration inside is increased.

  According to the method of the present invention, the types of peptides contained in the measurement sample can be greatly reduced, so that the condition setting for mass spectrometry can be simplified, and improvement in analysis accuracy can be expected. By analyzing the obtained peptide fragment, the antibody drug concentration in the blood can be quantified. Therefore, the method of the present invention can also be applied as a pretreatment method for antibody drug concentration measurement systems in preclinical and clinical trials.

  The method of the present invention can be applied not only to antibody drugs but also to many proteins. In addition to expanding the application in the pharmaceutical industry, it can be applied to basic research fields such as biomolecule interaction analysis. Can also be expected.

It is a conceptual diagram for demonstrating the principle of position-selective cutting by this invention. It is a schematic diagram for demonstrating the structure of an antibody. It is a schematic diagram showing one form of the peptide fragment preparation kit. It is an electrophoretic image of the quantity ratio examination experiment of protease. It is a mass spectrometry (MALDI-TOFMS) spectrum of the amount ratio examination experiment of protease. It is a mass spectrometry (MALDI-TOFMS) spectrum of a digestion time examination experiment. It is a database analysis result based on the mass spectrometry result of the trypsin digestion fragment of trastuzumab. It is a mass spectrometry (MALDI-TOFMS) spectrum of the trypsin digestion fragment of trastuzumab. It is a chromatogram of LS-MS measurement of a trypsin digested fragment of trastuzumab. 2 is a mass spectrometry (LS-MS) spectrum of a trypsin digested fragment of trastuzumab. It is a figure showing the amino acid sequence of the heavy chain (A) and light chain (B) of trastuzumab. The underline in the figure indicates peptide fragments identified by mass spectrometry of this experiment. It is an electrophoretic image of mixed protease digestion examination experiment. It is a mass spectrometry (MALDI-TOFMS) spectrum of mixed protease digestion examination experiment.

  In the method for preparing a peptide fragment of the present invention, the substrate protein to be cleaved is immobilized in the pores of the porous body, the porous body on which the substrate protein is immobilized, and the solid phase surface on which the protease is immobilized. Are brought into contact with each other. The contact between the two is performed, for example, in a liquid. FIG. 1 is a conceptual diagram for explaining the principle of protease digestion in the present invention.

In FIG. 1, fine particles 10 (average particle diameter D ₁ ) are shown as a solid phase, and protease 15 is immobilized on the surface thereof. The porous body 20 has a large number of pores 29 (average pore diameter D ₂ ), and the substrate protein 25 is immobilized in the pores. As described above, in the method of the present invention, both the protease 15 and the substrate protein 25 are fixed to a solid phase in a minute region, and protease digestion is performed by contact between the solid phases.

The average particle diameter D ₁ of the fine particles 10 is larger than the average pore diameter D ₂ of the porous body 20. Therefore, the fine particles 10 can access the vicinity of the surface layer of the pore 29, but cannot access the depth of the pore 29. Along with this, the protease 15 immobilized on the surface of the fine particles 10 is also inaccessible deep inside the pores 29. In FIG. 1, the dotted line near the pore 29 represents the boundary of the region accessible by the protease 15.

  Thus, the access of the protease 15 to the substrate protein 25 in the pore 29 is restricted in a position-specific manner, and the relative access probability to the liquid phase side of the substrate protein (the “Y” portion in FIG. 1). Is increased. Thus, the peptide fragment can be obtained by subjecting the substrate protein 25 to site-selective protease digestion.

[Substrate protein]
The substrate protein 25 is a protein to be analyzed. The type of the substrate protein is not particularly limited, but is preferably one having a molecular diameter larger than that of protease 15 from the viewpoint of performing site-selective cleavage. The substrate protein may be a protein complex. As for the molecular diameter, numerical values determined based on structural analysis by X-ray, NMR or the like can be obtained from various documents and databases. For example, the molecular diameter of the antibody is about 15 nm. Further, the molecular diameter can be obtained by X-ray small angle scattering or the like, and can be obtained roughly from the molecular weight. As a reference example, Table 1 shows the molecular weight and molecular diameter of a protein used as a marker molecule when examining the separation characteristics of an ultrafiltration membrane.

  The substrate protein 25 preferably binds site-specifically in the pores 29 of the porous body 20. By binding the substrate protein 25 in a site-specific manner, a site other than the binding site is selectively digested with protease. For example, proteins with tag sequences such as His tags (tag peptides consisting of about 6 consecutive histidine residues) or biotinylated peptides at the C-terminal or N-terminal, enzymes that specifically bind to specific substrates, etc. Can also be used as a site-specific binding protein.

  In the present invention, an antibody is particularly preferably used as a substrate protein that can bind site-specifically in the pores of the porous body. If the Fc domain of the antibody is immobilized on the porous body 20, the Fab domain can be selectively digested with protease. The type of antibody is not particularly limited, but a monoclonal antibody is preferable. Monoclonal antibodies include, for example, human antibodies such as panitumumab (Bectivix), ofatumumab (Arzela), golimumab (Simponi), ipilimumab (Yervoy); tocilizumab (Actemra), trastuzumab (Herceptin), bevacizumab (Avastin), omalizumab, omalizumab ), Mepolizumab (Bosatria), gemtuzumab ozogamicin (Myrotag), palivizumab (Sinazis), ranibizumab (Lucentis), sertolizumab (Simdia), ocrelizumab, mogamulizumab (Poterizio), eculizumab (Solitris), etc. Examples include chimeric antibodies such as rituximab (Rituxan), cetuximab (Arbitux), infliximab (Remicade), and basiliximab (Simlect). These are used as antibody drugs (molecular target therapeutic drugs), and it is necessary to quantify antibody concentrations in blood in clinical trials and the like.

  As will be described later in Examples, according to the method of the present invention, the Fab domain of a monoclonal antibody can be regioselectively digested with protease, and the resulting peptide fragment can be identified and quantified by mass spectrometry. Can be done. The analysis method of the present invention is a method for identifying (detecting) or quantifying an antibody by detecting a peptide fragment derived from a variable region of an antibody, and directly measuring an antibody-derived peptide fragment. Therefore, the analysis method of the present invention does not require a specific binding substance such as an antigen and can be applied regardless of the type of antibody. Therefore, the method of the present invention is not limited to the above-exemplified antibodies, and can be applied to newly developed monoclonal antibodies and the like.

[Porous material]
If the porous body 20 has many pores 29, the material will not be specifically limited. Although hemispherical pores are illustrated in FIG. 1, the shape of the pores is not particularly limited. Moreover, what formed the pore which penetrates a porous body like a porous film can also be used.

  As the porous body 20, activated carbon, a porous membrane, porous resin beads, metal particles, or the like can be used. Among these, those capable of selectively binding the above substrate protein are preferable, and those capable of binding the substrate protein in a site-specific manner are particularly preferable. For example, affinity column carrier beads used for purification of specific proteins or the like can satisfy such requirements.

  In the present invention, those in which linker molecules 21 that interact with the substrate protein 25 in a site-specific manner are immobilized in the pores 29 of the porous body 20 are preferably used. Examples of the interaction between the substrate protein and the linker molecule include chemical bond, hydrogen bond, ionic bond, complex formation, hydrophobic interaction, van der Waals interaction, electrostatic interaction, and stereoselective interaction. .

  The linker molecule is a functional group such as an amino group, a carboxyl group or an epoxy group; a labeled compound such as piotin or digoxigenin; a protein such as avidin, streptavidin protein A, protein G or immunoglobulin; various ligands; a substrate compound of enzyme; An optimal one can be appropriately selected from metal chelates and the like according to the type and binding site of the substrate protein.

  When the substrate protein 25 is an antibody, protein G, protein A or the like is preferably used as the linker molecule 21. Protein A and protein G bind site-specifically to the Fc domain of an antibody. By using the porous body 20 in which the linker molecule 21 such as protein A or protein G is immobilized in the pore 29, the Fc domain of the antibody (substrate protein 25) is immobilized in a site-specific manner in the pore. The Fab domain is located on the liquid phase side (near the surface layer of the pore). Thus, by immobilizing the antibody in the pore in a certain direction, the orientation of the antibody in the pore is controlled, and the position-selective digestion of the Fab domain by the protease becomes possible.

  In addition, by immobilizing the substrate protein in the pores and allowing it to exist in a fine environment such as the interface between the solid phase and the liquid phase, the substrate protein is easily denatured, the molecular fluctuations are perturbed, and the protease is attacked. Probability increases. Further, in the present invention, the protease is immobilized on the particles, so that it becomes an environment that is three-dimensionally stable and hardly undergoes self-digestion. Therefore, according to the method of the present invention, in addition to enabling regioselective protease digestion, high activity of the protease can be maintained.

The size of the pores 29 of the porous body 20 is not particularly limited. The size of the pore is such that, when the substrate protein 25 is immobilized, the tip of the substrate protein, that is, the site to be selectively digested, is located near the surface layer of the pore 29, etc. It is preferable to determine in consideration of the above. The average pore diameter _{D 2} of the porous body 20, for example, in the range of about 10 nm to 500 nm, and is set appropriately in a range smaller than the average particle diameter _{D 1} of the particles 10. The average pore diameter _{D 2} of the porous body 20, for example, preferably about 20 nm to 200 nm, about 30nm~150nm is more preferable. In particular, when the substrate protein 25 is an antibody, in order to immobilize the Fc domain in the pore and to selectively digest the Fab domain with protease, the pore diameter of the porous body is preferably 30 nm to 150 nm, and 40 nm. -120 nm is more preferable, and 50 nm to 100 nm is more preferable.

  The size of the linker molecule is selected so that the selective cleavage site of the substrate protein is located near the surface layer of the pore in consideration of the size of the pore and the substrate protein. The molecular size of the state in which the linker molecule and the substrate protein are bound is preferably about 0.5 to 1.5 times the pore diameter of the porous body, more preferably about 0.6 to 1.2 times, About 0.7 times to 1.1 times is more preferable, and about 0.8 times to 1 time is particularly preferable. In the case where the linker molecule is not fixed to the porous body 20 and the substrate protein is directly bound in the pores of the porous body, the molecular diameter of the substrate protein and the pore diameter of the porous body are as described above. It is preferable to satisfy the relationship.

[Immobilization of substrate protein]
The method for immobilizing the substrate protein 25 in the pores 29 of the porous body 20 is not particularly limited, and depends on the characteristics of the substrate protein and the porous body (or the linker molecule immobilized on the porous body). An appropriate method can be adopted. For example, when an antibody is immobilized on a porous body in which protein A or protein G is immobilized in the pores, a mixture of the porous body suspension and the antibody-containing solution is mixed into the pores. It is possible to easily immobilize the antibody.

  The amount ratio between the porous material and the substrate protein can be appropriately set according to the purpose. For example, when performing quantitative analysis of a substrate protein, it is desired that almost the entire amount of the substrate protein in the sample is immobilized on the porous body. Therefore, it is preferable to set the quantitative ratio so that the amount of the porous material is excessive with respect to the estimated content of the substrate protein in the sample.

[Protease]
Protease 15 recognizes the amino acid sequence of the substrate protein and selectively cleaves a specific bond of a specific sequence. In the present invention, the protease protein 15 cleaves the substrate protein 25 immobilized in the pores 29 of the porous body 20 at a specific amino acid sequence site to obtain a peptide fragment.

  Proteases include trypsin (which cleaves peptides at the C-terminal side of basic amino acid residues (Arg and Lys)), lysyl endopeptidase (which cleaves peptides at the C-terminal side of Lys residues), arginine endopeptidase (Arg). Cleavage peptide at C-terminal side of residue), chymotrypsin (cleave peptide at C-terminal side of aromatic amino acid residues (Phe, Tyr and Trp)), V8 protease (peptide at C-terminal side of Glu residue) Pepsin, papain and the like. In addition, protease can also be used in combination of 2 or more types.

  When the peptide fragment of the substrate protein after protease digestion is subjected to mass spectrometry as a measurement data, it is preferable to use a protease with less self-digestion and high selectivity of the cleaved sequence. When a commercially available protease is used, it is preferable to use a mass spectrometry grade or sequencing (sequence) grade protease. For example, native trypsin derived from living organisms is known to have low cleavage site specificity because it produces pseudotrypsin that exhibits chymotrypsin-like activity by autolysis. Therefore, as a mass spectrometry grade, a product obtained by reducing methylation of a lysine residue of trypsin to increase resistance to autolysis is commercially available.

  In order to increase the regioselectivity of the protease digestion of the substrate protein, it is important to limit the region where the protease can access the substrate protein. Therefore, the molecular diameter of the protease is preferably smaller than the molecular diameter of the substrate protein. More specifically, the molecular diameter of the protease is preferably 10 nm or less, more preferably 8 nm or less, further preferably 6 nm or less, and particularly preferably 5 nm or less. A protein having a molecular weight of about 30 kDa, such as trypsin and lysyl endopeptidase, has a molecular diameter of about 4 nm (see Table 1 above).

  Among the proteases, trypsin is particularly preferably used in the present invention. As described above, trypsin has a small molecular diameter and an active site exists in the molecule. Therefore, the region where the active site can access the substrate protein is limited, and the position selectivity of protease digestion can be enhanced. In particular, when the substrate protein is an antibody, trypsin is preferably used as a protease.

  In proteome analysis studies, mixed digestion with trypsin and lysyl endopeptidase has recently attracted attention as a technique for improving the recovery rate of peptide fragments (J. Proteome Res., 2012, 11 (11), 5145-5156). This is thought to be due to the fact that trypsin has a feature that the degradation reaction proceeds stepwise from the outside of the three-dimensional structure, whereas lysyl endopeptidase mainly cleaves the hinge region of the antibody first. On the other hand, in order to suppress cleavage of the antibody hinge region and selectively cleave the Fab domain (more preferably, the V region of the Fab domain), the present invention uses trypsin alone or lysyl endopeptidase. Even when these are used in combination, the amount of trypsin in the total protease is preferably 90% or more.

[Fine particles]
The fine particles 10 are used for the purpose of controlling protease access to the substrate protein 25 immobilized in the pores 29 of the porous body 20 by immobilizing the protease 15 on the surface thereof. Therefore, it is preferable that the average particle diameter D _{1 of} the fine particles 10 is larger than the average pore diameter D ₂ of the porous body 20 so that the fine particles 10 do not enter deeply into the pores 29 of the porous body 20. The average particle diameter _{D 1} of the particles 10 is more preferably 1.2 times or more the average pore diameter _{D 2} of the porous body 20, more preferably at least 1.5 times, particularly preferably at least 1.8 times.

  The shape of the fine particles 10 is not particularly limited, but spherical fine particles are preferable from the viewpoint of uniform protease access to the pores 29 of the porous body 20. The fine particles 10 preferably have a uniform average particle diameter.

If the average pore diameter of the porous body 20 is about 30 to 150 nm, the average particle diameter _{D 1} of the particles 10 is preferably at least 100 nm, more preferably not less than 150 nm. When the substrate protein 25 is an antibody and the average pore diameter of the porous body 20 is about 50 nm to 100 nm, the average particle diameter of the fine particles 10 is preferably 120 nm or more, more preferably 150 nm or more, and particularly preferably 170 nm or more. It is not particularly limited upper limit of the average particle diameter _{D 1} of the particles 10, in view of enhancing the digestion efficiency by proteases, preferably 1μm or less, more preferably 500nm or less, more preferably 300nm or less.

  The material of the fine particles 10 is not particularly limited as long as the protease can be immobilized on the surface, and a metal, a resin, or the like is appropriately used. Moreover, what coated the metal surface with resin, what coated the resin surface with the metal, etc. can also be used.

  The fine particles 10 are preferably those that can suppress the nonspecific protein adsorption to the surface and can selectively immobilize the protease. For example, as shown in FIG. 1, the surface of the fine particles specifically binds to the protease. The fine particles modified with the spacer 11 are preferably used. The spacer is preferably one that can bind to the protease and does not inactivate the protease.

  Further, from the viewpoint of controlling the access range of the protease 15 immobilized on the surface of the fine particles 10, the spacer 11 preferably has a small molecular diameter. The spacer molecular diameter is preferably 5 nm or less, more preferably 3 nm or less, and even more preferably 2 nm or less. The molecular weight of the spacer is preferably 2000 or less, more preferably 1500 or less, further preferably 1000 or less, and particularly preferably 800 or less. The spacer molecule capable of immobilizing protease with the above molecular diameter is preferably a non-protein, and a molecule having a functional group such as an amino group, an amide group, an ester group, an epoxy group, a carboxyl group, piotin, avidin, or chelate is preferable. For example, a spacer having an ester group is preferably used for fixing trypsin. In order to increase the efficiency of protease immobilization, molecules having an activated ester group are also preferably used as spacers.

  In the present invention, commercially available fine particles modified with spacer molecules can also be used. For example, as affinity purification fine particles, fine particles in which ester groups are modified with spacer molecules activated with N-hydroxysuccinimide are commercially available under the trade name “FG beads NHS”.

[Preparation of protease-immobilized microparticles]
The method for immobilizing the protease 15 on the surface of the fine particles 10 is not particularly limited, and an appropriate method can be adopted depending on the characteristics of the protease and the fine particles (or spacer molecules that modify the surface of the fine particles). For example, when trypsin is immobilized on the spacer-modified fine particle surface, the protease can be immobilized on the fine particle surface by mixing a suspension of fine particles and a solution containing trypsin.

  After immobilizing the protease on the surface of the microparticles, it is preferable to inactivate the active portion that is not bound to the protease on the microparticle surface. For example, if there is a spacer molecule on which the protease is not immobilized on the surface of the microparticles, the unbound spacer molecule binds to impurities in the sample and has an adverse effect on protease digestion or is produced by protease digestion. In some cases, the peptide fragments may be immobilized on the microparticles. Such imperfections are suppressed by blocking unbound spacers after immobilizing the protease. As a method for inactivating an active moiety that is not bound to a protease, chemical modification is preferred. For example, an activated ester group is inactivated by forming an amide bond by reaction with an amine.

[Protease digestion]
By contacting the porous body 20 on which the substrate protein 25 is immobilized and the microparticles 10 on which the protease 15 is immobilized on the surface in a liquid, the substrate protein is digested with the protease and peptide fragments are produced.

  The conditions for protease digestion in the present invention are not particularly limited, and conditions similar to those for general protease digestion can be appropriately employed. For example, it is preferable to incubate at a temperature of about 37 ° C. for about 4 to 20 hours in a buffer solution adjusted to near the optimum pH of the protease.

  The mixing amount ratio between the porous body on which the substrate protein is immobilized and the microparticles on which the protease is immobilized is not particularly limited, and may be set so that the amount of protease corresponds to the amount of the substrate protein. General protease digestion conditions are about substrate: protease = 100: 1 to 20: 1 (weight ratio). On the other hand, in the present invention, since the access between the substrate protein and the protease is physically restricted by the combination of the porous material and the fine particles, the amount of the protease is increased as compared with general protease digestion. Is preferred. For example, substrate protein: protease = about 30: 1 to 3: 1 is preferable, about 15: 1 to 4: 1 is more preferable, and about 10: 1 to 5: 1 is more preferable.

  In general, when selectively digesting an antibody in a biological sample such as blood with a protease, first, the protein G or the like-immobilized particles are mixed with the sample to immobilize the antibody on the particles, and impurities are removed. Thereafter, it is necessary to elute the antibody from the particles, denature the eluted antibody with urea or guanidine, and perform protease digestion. In contrast, in the method of the present invention, protease digestion is performed in a state where the antibody is immobilized on the porous body. In addition, since peptide fragments produced by protease digestion are present in the liquid phase, Fab domain peptide fragments can be obtained selectively without performing antibody elution or denaturation operations. Thus, according to the method of the present invention, peptide fragments can be recovered in a position-selective manner with a simpler operation than the conventional method.

[Peptide fragment preparation kit]
The peptide fragment preparation according to the present invention can also be prepared using a peptide fragment preparation kit prepared in advance. The peptide fragment preparation kit includes a porous body having pores capable of immobilizing a substrate protein and fine particles capable of immobilizing protease on the surface. The kit may further contain a protease. In addition, fine particles having a protease immobilized on the surface may be provided.

  FIG. 3 is a diagram showing an embodiment of a peptide fragment preparation kit. In FIG. 3, the fine particles 110 capable of immobilizing protease on the surface are provided as a suspension 131. The kit may further contain a protease. In addition, the microparticles 110 may be provided in a state where the protease is immobilized on the surface. The spin column 132 includes an inner container 135 and an outer container 136, which are formed in a detachable shape. A porous membrane 120 having pores capable of immobilizing a substrate protein is disposed at the bottom of the inner container 135. The porous membrane 120 has a pore diameter that does not allow liquid to permeate at normal pressure.

  When peptide fragments are prepared using this spin column, first, a sample containing a substrate protein (for example, a specimen such as blood) is placed in the inner container 135 of the spin column, and the sample and the porous membrane are brought into contact with each other. . In order to bring the sample and the porous membrane into uniform contact, the container may be shaken as necessary. By this operation, a substrate protein such as an antibody is immobilized in the pores of the porous membrane 120.

  The sample liquid after the substrate protein is immobilized on the porous membrane is preferably discharged from the inner container 135. The liquid may be discharged from the opening of the inner container by an operation such as pipetting, or the liquid may be discharged from the bottom of the inner container through a porous membrane by centrifugation or the like. Thereafter, cleaning is performed with an appropriate solution as necessary.

  In the inner container 135 in which the substrate protein is immobilized on the porous membrane 120, the fine particles 110 in which the protease is immobilized on the surface are added. As described above, the protease may be provided in a state of being immobilized on the microparticles in advance, or the protease can be immobilized on the surface of the microparticles immediately before use.

  For the purpose of optimizing protease digestion conditions, a solution such as a buffer may be further added as necessary. The substrate protein immobilized on the porous membrane 120 in the inner container is digested by the protease immobilized on the surface of the fine particles 110. The conditions for protease digestion can be appropriately set as described above. Peptide fragments produced by protease digestion migrate into the liquid phase.

  By recovering the liquid phase after protease digestion, a peptide fragment in which the substrate protein is cleaved regioselectively is obtained. The recovery method of the liquid phase is not particularly limited, but a method of recovering from the bottom of the inner container 135 into the outer container 136 through a porous membrane is easier than centrifugation. Thereafter, for the purpose of elution of peptide fragments retained in the pores of the porous membrane, operations such as washing and elution may be performed.

  Thus, by using the kit, the operation of preparing the peptide fragment according to the present invention can be simplified, and automation by the apparatus can be easily achieved. In particular, trypsin and the like can retain activity even when immobilized on the surface of the microparticles. Therefore, if the protease is provided as a component of the kit in a state of being immobilized on the surface of the microparticles, the operation of preparing the peptide fragment can be further performed. It can be simplified.

[analysis]
By analyzing the sample containing the peptide fragment obtained above by chromatography or mass spectrometry, the substrate protein can be identified and quantified. In the present invention, since the substrate protein is subjected to protease treatment in a regioselective manner, the types of peptide fragments contained in the sample are reduced. Therefore, analysis conditions can be easily set by mass spectrometry or the like. In the analysis, if necessary, the sample may be used for the analysis after treatment such as desalting, solubilization, extraction, concentration, and drying.

  Mass spectrometry is suitable for identifying and quantifying substrate proteins from peptide fragments produced by protease digestion. Since mass spectrometry can determine an amino acid sequence, it can be determined whether or not a peptide fragment is a peptide fragment derived from a specific protein such as an antibody. Further, the concentration of the peptide fragment in the sample can be determined based on the peak intensity.

The ionization method in mass spectrometry is not particularly limited. Electron ionization (EI) method, chemical ionization (CI) method, field desorption (FD) method, fast atom collision (FAB) method, matrix-assisted laser desorption ionization (MALDI) For example, an electrospray ionization (ESI) method may be employed. The analysis method of the ionized sample is not particularly limited, and a magnetic field deflection type, a quadrupole (Q) type, an ion trap (IT) type, a time of flight (TOF) type, a Fourier transform ion cyclotron resonance (FT-ICR) type. Etc. can be appropriately determined according to the ionization method. Further, MS / MS analysis or multi-stage mass analysis of MS ³ or higher can be performed using a triple quadrupole mass spectrometer or the like.

For the purpose of ensuring more reliable separation of peptide fragments and improving analytical accuracy, the sample before being subjected to mass spectrometry can be separated and concentrated by liquid chromatography (LC), solid phase extraction (SPE), etc. Good. When separating a sample by LC, LC / MS provided with LC as a pre-stage of mass analysis may be used, and the eluate from LC may be directly ionized and subjected to mass analysis. Analysis can also be performed by LC / MS / MS or LC / MS ⁿ combining LC and tandem mass spectrometry. Alternatively, the eluate from LC may be collected once and then subjected to mass spectrometry. LC column and SPE carrier are not particularly limited, and a hydrophobic column such as C30, C18, C8, C4 or the like generally used for peptide analysis, a carrier for hydrophilic affinity chromatography, etc. are appropriately selected. Can be used.

  Existing databases can also be used to identify proteins such as antibodies based on mass spectrometry results. In the present invention, peptide fragments obtained by subjecting a substrate protein such as an antibody to position-specific protease digestion are used, so that the hit rate and data accuracy by database search can be improved. In addition, the substrate protein can be identified by specifying the amino acid sequence of the peptide fragment by multistage mass spectrometry or the like. For example, when the substrate protein is an antibody, the antibody can be identified by determining the amino acid sequence of a peptide fragment containing at least a part of the amino acid sequence of the complementarity determining region (CDR) having an amino acid sequence specific to the antibody.

  In addition, when performing antibody detection or quantification based on the detection result of a specific peptide fragment containing a CDR sequence, the number of amino acid residues of the peptide to be detected is preferably about 5 to 30, and about 7 to 25 More preferred. If the number of amino acid residues is excessively small, it is difficult to distinguish from contaminants and peptide fragments derived from other parts of the same protein, which may cause false detection. On the other hand, if the number of amino acid residues is excessively large, detection may be difficult or the quantitative property may be lowered due to reasons such as difficulty in ionization.

  When quantifying the concentration of substrate protein, calculate the amount of substrate protein based on the peak area and peak intensity of the detected peptide fragment ions (in the case of multi-step MS, cleavage ions obtained by cleavage of peptide fragment ions). it can. For example, peptide fragments in a sample can be obtained by associating a calibration curve (calibration curve) obtained in advance with a peak area, or by associating a peak area derived from an internal standard added to a sample with a peak area derived from the sample. The amount and concentration of the substrate protein are calculated based on the peptide fragment concentration.

  As described above, according to the present invention, both a substrate protein and a protease are immobilized on a solid phase, and the access to both is physically controlled, whereby a specific site of the substrate protein is selectively digested with protease. can do. The obtained peptide fragment can be analyzed by a known method such as mass spectrometry, and the protein in the sample can be identified and quantified without going through complicated steps.

  The method of the present invention is particularly suitable for the detection and quantification of antibodies. A peptide containing the amino acid sequence of the complementarity determining region is obtained by selectively digesting the Fab region with a protease and mass spectrometry of the obtained peptide fragment sample. The sequence and amount of fragments can be determined. In addition, the method of the present invention is simple in operation, can ensure reproducibility and quantification, and can also be automated, so pharmacokinetic analysis, interaction analysis using antigen-antibody reaction, various interactomes It can also be applied to basic research such as analysis and identification of immunoprecipitated proteins. In addition, it can be expected to be applied to sequence analysis of biomolecular drugs such as antibody drugs, quality assurance, and confirmation of the identity of generic drugs.

  In the following, an experimental example is shown in which human immunoglobulin G (IgG) and trastuzumab (trade name: Herceptin) are digested with protease by the method of the present invention, and the obtained peptide fragment sample is subjected to mass spectrometry. In addition, this invention is not limited to the following examples.

Hereinafter, the description of% represents% by weight unless otherwise specified. The reagents used in this experimental example are as follows.
Trypsin (sequence grade, promega)
Lysyl end peptider (mass spectrometry grade, Wako Pure Chemical Industries)
2-morpholinoethanesulfonic acid (MES, Doujin Chemical)
2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES, Doujin Chemical)
Tris (hydroxymethyl) aminomethane (Tris, Wako Pure Chemical Industries)
Organic solvents other than those described above were obtained from Wako Pure Chemical Industries unless otherwise specified.

Moreover, each buffer used the following buffer solutions which adjusted pH using the precision pH meter.
MES buffer: 25 mM MES-NaOH, pH 5.5
HEPES buffer: 25 mM HEPES-NaOH, pH 7.0
Ethanolamine buffer: 1M ethanolamine-HCl, pH 8.0
Tris buffer: 25 mM Tris-HCl, pH 8.0

<Preparation of antibody-immobilized porous body>
In 200 μL of MES buffer, 5 μL of a suspension of resin beads (Pierce Biotechnology, Protein G UltraLink resin, average particle size: 100 μm, pore size: 50 to 100 nm) with Protein G bound to the surface of the porous beads is added. After adding the antibody solution to the solution, the mixture was slowly stirred at room temperature for about 1 hour, and the antibody was bound to Protein G on the surface of the resin beads to be immobilized. Thereafter, the resin beads are precipitated by centrifugation (15000 rpm, 1 minute) at 4 ° C. After removing the supernatant, washing with the Tris buffer and centrifugation are repeated twice, and the porous beads are suspended in the Tris buffer (200 μL). Made cloudy. As the antibody solution, a human immunoglobulin (IgG) solution (10 mg / mL, Sigma-Aldrich) and a trastuzumab (Herceptin, 20 mg / mL, Chugai Pharmaceutical) solution were used.

<Preparation of protease-immobilized microparticles>
The surface of fine particles having an average particle diameter of 190 nm was modified with a spacer in which a carboxy group was activated with N-hydroxysuccinimide (see the following chemical formula (L is a binding site to the fine particle surface), spacer length 1 nm). Nanoparticles for immobilizing protease (Tamakawa Seiki, FG beads NHS) were used.

  Centrifugation (15000 rpm, 5 minutes) of isopropanol suspension of FG beads at 4 ° C. was allowed to settle, and the supernatant was removed, followed by washing with methanol. A solution containing 50 μg of protease dissolved in 200 μL of HEPES buffer was added to the above microparticles to suspend the microparticles. During suspension, ultrasonic treatment was performed for several seconds so that the temperature of the suspension did not increase.

  The fine particle suspension was stirred at 4 ° C. for 30 minutes, and centrifuged at 4 ° C. (15000 rpm, 5 minutes) to precipitate the fine particles. After removing the supernatant, 200 μL of ethanolamine buffer was added to suspend the beads. The mixture was made turbid and stirred at 4 ° C. for 30 minutes to block excess N-hydroxysuccinimide groups on the surface of the fine particles with ethanolamine. Thereafter, centrifugation was performed at 4 ° C. (15000 rpm, 5 minutes) to precipitate the microparticles, and after removing the supernatant, washing with Tris buffer and centrifugation were repeated twice and suspended in Tris buffer (100 μL). The protease concentration in this suspension is 0.5 μg / μL.

[Experiment 1: Confirmation of amount of antibody immobilized on porous material]
In preparation of the antibody-immobilized porous body, the amount of Protein G-bound resin bead suspension for 100 μg of IgG was changed in the range of 0 to 20 μL, and the supernatant was analyzed by SDS-PAGE electrophoresis. From the number of band pixels, the approximate amount of unbound IgG remaining in the supernatant (remaining antibody amount) was determined. As the amount of Protein G binding resin beads increases, the remaining amount of antibody tends to decrease. When the amount of Protein G binding resin beads is 10 μL, the remaining amount of antibody is about 3%. It was confirmed that the catalog specs were almost reproduced (data not shown).

[Experiment 2: Examination of the ratio of antibody to protease]
The IgG-immobilized porous body suspension (Protein G-IgG) and protease-immobilized microparticles (FG beads-Tripsin) were mixed, and protease digestion was performed while slowly stirring at 37 ° C. for 15 hours. Thereafter, the resin was precipitated by centrifugation (15000 rpm, 5 minutes) at 4 ° C., and the liquid phase (supernatant) was collected. The above experiment was performed by changing the amount of protease-immobilized microparticles so that the amount of protease was 5 μg (level 1), 10 μg (level 2), and 25 μg (level 3). In Levels 4 to 6, a similar experiment was performed using a porous body (Protein G UltraLink resin) in which IgG was not immobilized as it was instead of the IgG-immobilized porous body suspension. In Levels 7 and 8, only the protease-immobilized fine particles (FG beads-Tripsin) were incubated at 37 ° C. for 15 hours without using a porous material.

  Table 2 shows the level of each experiment. The weight (μg) in Table 2 is the amount of protein (IgG or trypsin) in the sample. The SDS-PAGE electrophoresis image of the obtained supernatant is shown in FIG. In FIG. 4, the leftmost lane is a molecular weight marker.

(Mass spectrometry)
The supernatants of the above levels 1 to 6 were analyzed by MALDI-TOFMS (Shimadzu Corporation, AXIMA Resonance MALDI-QIT TOF MS). First, trifluoroacetic acid was added to 20 μL of the supernatant to a final concentration of 0.5%, and purification was performed using a hydrophobic resin-filled chip (Millipore, ZipTip uC18), and then elution was performed twice with 1 μL. Went. The eluate was directly applied on a MALDI stainless steel target and air-dried in a clean bench. After air drying, 1 μL of a 10 mg / mL 2,5-dihydroxybenzoic acid solution (DHBA, Shimadzu GLC, water / acetonitrile = 50/50) was overlaid, and mass spectrometry was performed. In addition, m / z of the apparatus was performed using Angiotensin II peptide (m / z = 1046.54, Sigma-Aldrich) and ACTH fragment peptide (m / z = 2465.20, Sigma-Aldrich). The MS spectrum is shown in FIG.

  The bands of levels 4 to 6 in FIG. 4 were all the same as the bands of levels 7 and 8. In addition, the peaks at m / z = 842, 1045, 2211 and 2283 detected at levels 4 to 6 in FIG. 5 are all trypsin self-digesting fragments. From these results, it is understood that Protein G is not digested even when the protease-immobilized microparticles and the Protein G porous body are brought into contact with each other. This is probably because trypsin immobilized on the surface of the fine particles cannot access Protein G in the fine pores because the pore size of the porous material is smaller than the particle size of the protease-immobilized fine particles.

  In levels 1 to 3 in FIG. 5, in addition to the trypsin self-digesting fragment, peaks at m / z = 835, 913, 1187, 1287, 1510, 1678, and 1946 were detected. It was confirmed that. From this result, it was shown that IgG can be selectively digested without contacting protein G of the porous material by contacting the porous material immobilized with IgG and the microparticles immobilized with trypsin. .

  When the levels 1 to 3 in FIG. 4 are compared, it can be seen that the band on the polymer side decreases as the amount of trypsin increases, and the antibody digestion reaction proceeds well. On the other hand, self-digestion has become prominent with the increase in trypsin content. Based on these results, level 2 (substrate: enzyme ratio = 10: 1) was set as the standard condition, and the following conditions were examined.

  General protease digestion conditions are substrate protein: protease = 100: 1 to 20: 1. In this method, since the access between the substrate protein and the protease is physically restricted by the combination of the porous material and the fine particles, the amount of the protease is larger than the general protease digestion, and the optimum substrate enzyme The ratio is estimated to be about 10: 1 to 5: 1.

[Experiment 3: Evaluation of recovered peptide against digestion time]
Condition 2 in Experiment 2 above, ie, an IgG-immobilized porous body suspension (IgG solid phase amount 100 μg) and protease-immobilized microparticles (trypsin solid phase amount 10 μg) were mixed and digestion time at 37 ° C. (1) 15 minutes, (2) 45 minutes, (3) 90 minutes, (4) 180 minutes, (5) 360 minutes, (6) 15 hours (overnight, O / N). Except that, trypsin digestion was performed in the same manner as in Experiment 2, and the obtained sample was subjected to mass spectrometry. The MS spectrum is shown in FIG.

  As shown in FIG. 6, it can be seen that the peptide fragment peak increases with increasing digestion time, and the peptide fragment recovery amount increases. When (5) 360 minutes and (6) all night are compared, the recovery rate is higher overnight, and in particular, the accumulation of fragments that are susceptible to protease digestion such as m / z = 1187, 1510, 1678 becomes more prominent. There was a trend. However, these fragments are derived from the C region of the antibody and do not contribute to the analysis of peptide fragments containing CDRs. Therefore, the protease digestion time was set to 6 hours, and the following examination was performed.

[Experiment 4: Trypsin digestion and mass spectrometry of trastuzumab]
An antibody-immobilized porous material is prepared using Herceptin instead of IgG as an antibody, and the conditions selected in Experiments 2 and 3 above, ie, the amount of protease-immobilized fine particles with respect to 100 μg of antibody solid phase: 10 μg, digestion time: 6 hours Digestion of trypsin was performed under the conditions described above, mass spectrometry was performed, and database (Mascot server) analysis was performed based on the mass spectrometry results. As shown in FIG. 7, Herceptin (Chain B, X-Ray Structures Of The Antigen-Binding Domains From Three Variants Of Humanized Anti-P185-Her2 Antibody 4d5 And Comparison With Molecular Modeling) is identified with a very high score. I understand.

  In this experiment, more detailed analysis was performed to confirm that the peptide fragment of Herceptin was detected by mass spectrometry. FIG. 8 represents a mass spectrometry (MALDI-TOFMS) spectrum.

  In addition, the supernatant after digestion was analyzed by LC-MS (Shimadzu Corporation, LCMS-8080 Triple-quadrupole ultra high-performance liquid chromatography-MS). The chromatogram of LC-MS is shown in FIG. 9, and the MS spectrum is shown in FIG. In the measurement of LC-MS, the supernatant was added with formic acid so that the final concentration was 0.5%, and the LC conditions were as follows.

<HPLC solution>
Solution A: 0.1% formic acid, 1% acetonitrile / water solution Solution B: 0.1% formic acid, acetonitrile solution <column>
ShimPack ODS XR-ODS II (Inner diameter 2mm, Column length 50mm)
Column temperature: 40 ° C
Flow rate: 0.4 mL / min Injection volume: 20 μL
<Gradient program>
0-2 minutes:% B = 0
2-10 minutes:% B = 0-40 gradient 10-11 minutes:% B = 40-98 gradient 11-13 minutes:% B = 98
13-13.5 min:% B = 98-0 Gradient 13.5-15 min:% B = 0

  Peptide sequence portions detected and identified by mass spectrometry in the heavy chain of trastuzumab (FIG. 11 (A), SEQ ID NO: 1 in the sequence listing) and light chain (FIG. 11 (B), SEQ ID NO: 2 of the sequence listing) Is underlined. As shown in FIG. 11A, all of heavy chain CDR1 (SEQ ID NO: 3 in the sequence listing), CDR2 (SEQ ID NO: 4 in the sequence listing), and CDR3 (SEQ ID NO: 5 in the sequence listing) have been detected. I understand. Further, as shown in FIG. 11 (B), CDR1 (SEQ ID NO: 6 in the sequence listing) and CDR2 (SEQ ID NO: 7 in the sequence listing) are detected in the light chain. In light chain CDR3 (SEQ ID NO: 8 in the sequence listing), the length of a trypsin digested fragment containing this sequence is 4 amino acid residues or 37 amino acid residues, and a fragment suitable for mass spectrometry could not be obtained. Although it could not be identified, it is considered that a peptide fragment capable of detecting CDR3 of the light chain by mass spectrometry can be prepared by changing the type of protease. In addition, in this experiment, CDR3 of the light chain could not be detected, but 5 of the total of 6 CDRs of heavy chain and light chain were detected, so that trastuzumab was identified by database analysis as shown in FIG. Has been.

  As described above, since the peptide fragment prepared by the method of the present invention is obtained by subjecting the antibody to position-specific protease digestion, the antibody is identified by mass spectrometry measurement that does not require setting of complicated measurement conditions. I understand that I can do it.

[Experiment 5: Examination of mixed protease digestion]
In the following, in order to examine the applicability of mixed protease digestion in the system of the present invention, a protease digestion experiment was conducted in a combined system of trypsin and lysyl endopeptidase (Lys-C).

  As in the above experimental examples, an antibody-immobilized porous material in which 100 μg of antibody (IgG or Herceptin) was immobilized on Protein G, which is a porous material, and microparticles in which 10 μg of protease were immobilized were incubated at 37 ° C. for 6 hours. Stirring and protease digestion of the antibody was performed. The ratio (weight ratio) of trypsin to lysyl endopeptidase was (1) 10: 0, (2) 9: 1, and (3) 8: 2 when IgG was used as an antibody and when Herceptin was used. (4) The experiment was conducted at 0:10, and each of the digested supernatant and the immobilized component on the surface of the porous body was analyzed by SDS-PAGE electrophoresis. An electrophoretic image is shown in FIG. In FIG. 12, the leftmost lane is a molecular weight marker.

  Mass spectrometry was performed on the supernatant after digestion at the above levels 1 to 4 when Herceptin was used as an antibody in the same manner as in Experiment 2 above. The MS spectrum is shown in FIG. Further, based on the mass analysis results, database analysis by Mascot server was performed in the same manner as in Experiment 4. Level 1 (100% trypsin) is the same as in Experiment 4 (FIGS. 7 and 8). The results of database analysis of levels 2 to 4 were as follows (data not shown)

<Level 2: Trypsin: Lysyl endopeptidase = 90: 10>
Mixture
gi | 442924 Mass: 23708 Score: 117 Expect: 4.9e-007 Matches: 13
Chain B, X-Ray Structures Of The Antigen-Binding Domains From Three Variants Of Humanized Anti-P185-Her2 Antibody 4d5 And Comparison With Molecular Modeling
gi | 184747 Mass: 36012 Score: 64 Expect: 0.11 Matches: 10
immunoglobulin gamma-1 heavy chain constant region [Homo sapiens]

<Level 3: Trypsin: Lysyl endopeptidase = 80: 20>
Mixture
gi | 442924 Mass: 23708 Score: 115 Expect: 7.8e-007 Matches: 13
Chain B, X-Ray Structures Of The Antigen-Binding Domains From Three Variants Of Humanized Anti-P185-Her2 Antibody 4d5 And Comparison With Molecular Modeling
gi | 184747 Mass: 36012 Score: 54 Expect: 1.1 Matches: 9
immunoglobulin gamma-1 heavy chain constant region [Homo sapiens]

<Level 4: Lysyl endopeptidase 100%>
gi | 184747 Mass: 36012 Score: 71 Expect: 0.021 Matches: 8
immunoglobulin gamma-1 heavy chain constant region [Homo sapiens]

  In Levels 2 and 3 in which trypsin and lysyl endopeptidase were used in combination, the analysis results showed a constant region of a human-derived antibody in addition to the antigen-binding region of Herceptin. Moreover, in the level 4 which used only a lysyl endopeptidase, the analysis result that Herceptin was not contained and only the constant region of the antibody was detected preferentially was shown.

  In the electrophoretic image of FIG. 12, as the lysyl endopeptidase ratio increases, the number of peptide fragments in the supernatant tends to increase and the band area tends to increase, and the digestion efficiency and peptide recovery rate increase. Recognize. Moreover, also in FIG. 13, the kind and quantity of the peptide fragment detected are increasing with the raise of the ratio of a lysyl endopeptidase. However, many of the peptide fragments newly detected at levels 2 to 4 are derived from the Herceptin Fc domain, and the position selectivity of protease digestion (characteristic for selectively digesting the Fab domain) is reduced. Recognize.

  From these results, as the use amount of lysyl endopeptidase increases, the digestion efficiency of the antibody is improved, the regioselectivity of protease digestion is lowered, and the production amount of peptide fragments in the constant region increases. It can be seen that the relative production amount of peptide fragments in the region tends to decrease, so that the detection and identification accuracy of the antibody tends to decrease. Therefore, it is preferable to use trypsin alone, and trypsin and lysyl endopeptidase are used in combination from the viewpoint of carrying out position-selective cleavage of the Fab region of the antibody and specific detection of CDR by the method of the present invention. In this case, it can be said that the amount of lysyl endopeptidase mixed is preferably 10% or less.

  In Level 4, which used only lysyl endopeptidase, site-specific digestion was not performed, whereas in Level 1, which used only trypsin (Experiment 4), CDR was efficiently detected by database analysis. This indicates that the V domain of the Fab domain is selectively digested with protease. According to the above results, when the present invention is applied using trypsin when the substrate protein is an antibody, the three-dimensional access of the protease to the antibody is appropriately controlled, and position-selective protease digestion is possible. It can be said that there was.

  In addition, this experiment showed that when trypsin and lysyl endopeptidase were used in combination, each protease could cleave the substrate protein immobilized in the pore without losing its function. The antibody has a tendency that the position specificity decreases when the amount of lysyl endopeptidase is increased because the V region is present at the tip of the molecule. However, when other proteins are used as a substrate, a combined protease system is used. This suggests the possibility of improving the position selectivity and digestion efficiency.

  As described above in each experimental example, according to the present invention, a peptide fragment sample can be obtained by subjecting a protein such as an antibody to site-selective protease digestion by a simple method. It can be seen that it is suitable for protein identification and detection by analysis.

DESCRIPTION OF SYMBOLS 10 Fine particle 11 Spacer 15 Protease 20 Porous body 21 Linker molecule 25 Substrate protein 29 Pore 110 Fine particle 120 Porous membrane 131 Fine particle suspension 132 Spin column 135 Inner container 136 Outer container

Claims (13)

Immobilizing a substrate protein to be cleaved in at least one pore of a porous body; and a substrate protein immobilized in the pore and a protease immobilized on a solid phase surface, Contacting a protease to access a cleavage site of the substrate protein, and cleaving the substrate protein at the cleavage site;
A method for preparing a peptide fragment.   The method for preparing a peptide fragment according to claim 1, wherein the solid phase surface is a surface of at least one fine particle.   The peptide fragment preparation according to claim 2, wherein the surface of the microparticle is modified with a spacer molecule capable of binding to the protease, and the protease is immobilized on the surface of the microparticle through the spacer molecule. Method. The at least one pore is a plurality of pores;
The at least one fine particle is a plurality of fine particles;
The method for preparing a peptide fragment according to claim 2 or 3, wherein an average particle diameter of the plurality of microparticles is larger than an average pore of the plurality of pores.   The method for preparing a peptide fragment according to claim 4, wherein the porous body has an average pore diameter of 30 to 150 nm, and the fine particles have an average particle diameter of 100 nm or more.   The method for preparing a peptide fragment according to any one of claims 1 to 3, wherein the contact between the substrate protein and the protease is carried out in a liquid.   The method for preparing a peptide fragment according to any one of claims 1 to 6, wherein in the immobilization of the substrate protein, a site different from the cleavage site of the substrate protein is immobilized in the pore. A linker molecule that interacts with the substrate protein in a site-specific manner is immobilized in the pores,
The method for preparing a peptide fragment according to any one of claims 1 to 7, wherein in the immobilization of the substrate protein, the substrate protein is immobilized in the pore through the linker molecule.   The molecular size of the state in which the linker molecule and the substrate protein are bound after the substrate protein is immobilized is 0.5 to 1.5 times the average pore diameter of the porous body. A method for preparing the described peptide fragment.   The method for preparing a peptide fragment according to any one of claims 1 to 9, wherein the substrate protein is an antibody.   The method for preparing a peptide fragment according to claim 10, wherein the antibody is a monoclonal antibody. In immobilization of the substrate proteins, Fc domains of the antibody is immobilized in the pores,
The method for preparing a peptide fragment according to claim 10 or 11, wherein the Fab domain of the antibody is regioselectively cleaved by the protease in the cleavage of the substrate protein.   The method for preparing a peptide fragment according to any one of claims 1 to 12, wherein the protease is trypsin.