JP2007309689A - Identification technique for protein binding region - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for efficiently identifying a protein binding region in the interaction between proteins like antigen-antibody reaction. <P>SOLUTION: The first protein of first and second protein interacting with each other is fixed on a support, the second protein is fragmented to be added to the first protein, the second protein fragments captured by the first protein are separated and the amino acid sequence of the separated second protein fragments is analyzed by mass analysis to identify the binding region of the second protein to the first protein. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a technique for identifying a binding site of a protein that interacts with each other such as an antigen and an antibody. More specifically, the present invention relates to a technique for identifying a binding site by allowing a first protein immobilized on a support to capture a fragmented second protein and analyzing the fragment.

  With the accumulation of a large amount of genomic information, the life science field is shifting from genomics to proteomics. Since proteins exert their functions by interacting with other molecules, elucidation of protein interaction networks is important for comprehensive understanding of life phenomena. In addition, it has been clarified that abnormalities in protein-protein interactions are associated with various diseases. For example, allergy, which is an autoimmune disease, is caused by excessive reaction of IgE, which is one of antibodies against allergens. Accordingly, development of drugs capable of controlling such interactions as therapeutic agents for allergies is underway.

  In a conventional allergy test, the blood concentration of an IgE antibody specific for an allergen is measured, and if the IgE amount is greater than or equal to a threshold value, an allergy is diagnosed. An effective treatment for allergies is a technique that suppresses excessive reactions by inoculating a vaccine, that is, an attenuated allergen in advance.

  These tests and treatments often use allergens extracted from natural products. However, allergens derived from natural products have a problem that the quality is easily changed for each production batch. Also, it cannot be determined which part of the allergen IgE recognizes. Furthermore, natural product-derived allergens are mostly a mixture of a plurality of proteins and low-molecular compounds, and to which molecule allergies occur varies depending on the patient. Therefore, when an allergen corresponding to the vaccine is not contained, the vaccine has no effect, and conversely, it may lead to another side effect such that another allergy is caused by an unrelated molecule.

  In order to improve the above problems, the use of recombinant protein allergens has been proposed. By using the purified recombinant protein for the test, it becomes possible to detect only allergy to the protein. Furthermore, when using the same protein as a vaccine, attention has been focused on techniques for enhancing the effect and reducing side effects by artificially introducing deletions and mutations. For this purpose, it is necessary to know in advance which site in the allergen molecule the antibody recognizes (Non-patent Document 1).

  There are many protein-protein interactions involved in important biological functions and diseases regardless of allergen-antibody. Identification of these binding sites is important in elucidating disease mechanisms and developing drugs that inhibit interactions.

As a method for identifying a protein binding site, many binding site mapping methods using peptide arrays have been reported. In this method, a device in which a peptide containing a part of the amino acid sequence of the target protein is artificially synthesized and arrayed on a support is used. A recognition site can be determined by adding a sample on the array and examining which peptide reacts with it (Non-Patent Document 2). However, the production of a peptide array is costly, and it is impossible to handle all of the myriad test objects. On the other hand, methods using mass spectrometry (Patent Documents 1 and 2) have also been reported, but these are merely for the purpose of ligand search, and do not enable the determination of the binding site sequence.
Japanese translation of PCT publication No. 2002-501176 JP-T-2004-534519 Methods. 2004 Mar; 32 (3): 313-20. Review. Curr Opin Biotechnol. 2001 Feb; 12 (1): 59-64.

  An object of the present invention is to provide a technique for efficiently identifying a binding site in a protein-protein interaction such as an antigen-antibody reaction.

  In order to solve the above-mentioned problems, intensive studies were conducted, one protein was immobilized on a support, the other fragmented protein was captured here, and the captured fragment was analyzed by MS / MS. We succeeded in identifying the binding site between.

  That is, the present invention is a method for identifying a binding site between interacting proteins, wherein the first protein is immobilized on a support, the second protein is fragmented, and added to the first protein. , Separating the second protein fragment captured by the first protein, analyzing the amino acid sequence of the separated second protein fragment by mass spectrometry, and analyzing the first protein of the second protein. A method of identifying a binding site for a protein is provided.

  Examples of interacting proteins include receptors (first proteins) and their ligands (second proteins), antibodies (first proteins) and various antigens (second proteins) including allergens. Can do. The second protein is not limited to one type and may be two or more types.

  The immobilization method of the first protein is not particularly limited, and is selected according to the kind and purpose of the protein such as physical adsorption, hydrophobic bond, chemical bond, and covalent bond.

  In the immobilization, it is important to maintain the higher-order structure of the first protein in the immobilization and its orientation (make the binding site face outward). For example, when there are a plurality of binding sites or when the binding sites are unknown, it is preferable to use physical adsorption capable of random adsorption. On the other hand, when the first protein is an antibody, it is desirable to immobilize the antigen binding site outward by immobilizing the N-terminus of the antibody on the support via an appropriate linker. An example of such a linker is ProLinker (registered trademark), which is a kind of calix crown derivative. If necessary, the first protein may be used after being denatured.

  The support is not particularly limited as long as the protein to be used can be immobilized. For example, membranes such as nitrocellulose and PVDF, silicon such as glass and wafer, resins such as plastic, and metals can be used. The support may be subjected to a surface treatment (modification) suitable for immobilization. Examples of such surface treatment (modification) include poly-L-lysine and aminosilane that can immobilize the target molecule by physical adsorption, and aldehyde groups and epoxy groups that can immobilize the target molecule by covalent bonds. Avidin, Ni-NTA, and the like that can be immobilized by utilizing an affinity with a functional group and a target molecule can be used. A solid phase composed of a thin layer of a hydrophilic porous matrix such as polyacrylamide gel or agarose gel can also be used.

  Examples of the method for fragmenting the second protein include a chemical fragmentation method and an enzymatic method. Examples of the former include the cyanogen bromide method, and examples of the latter include digestion with trypsin, chymotrypsin, ArgC, LysC, AspN, V8 (GlcC) and the like.

  For fragmentation, it is also effective to combine a plurality of types of fragmentation methods. For example, the following examples can be considered as a method for selecting a combination. First digest with easy-to-obtain and highly specific trypsin. If trypsin is insufficiently digested (fragment is too large for MS / MS analysis), add AspN or V8 with a different recognition sequence from trypsin. If digestion is still insufficient, change to chymotrypsin with low specificity and relatively random digestion. If digestion is excessive with trypsin (the binding site is digested and cannot be identified), the enzyme is changed to ArgC or LysC, which has fewer recognition sequences than trypsin. Change to the chemical digestion method for proteins that are difficult to undergo enzymatic digestion.

  Finally, the second protein is preferably fragmented to a molecular weight of 4000 or less (about 40 amino acids), particularly 2000 (about 20 amino acids) or less. Fragmentation can be confirmed by SDS-PAGE, HPLC, and the like.

The second protein fragment captured by the first protein is separated by elution using an appropriate solvent. For the elution, a method generally used for protein dissociation is used. For example, elution with acids such as 0.1M glycine buffer (pH 2), denaturants such as 8M urea, 6M guanidine and 3% SDS, chaotropic ions such as 4.5M MgCl _{2 and} nonpolar solvents such as 50% ethylene glycol That's fine.

  Samples containing the eluted fragments need to be desalted before being subjected to mass spectrometry. Desalting devices include columns filled with reversed-phase resins such as C18 (PIERCE, PepClean, etc.), cartridges (3M, Empore extraction disks, etc.), pipette tips (Millipore, ZipTip, etc.), multi-well filters Plates (Millipore, ZipPlatemaikuro-SPE plate, etc.) are used.

  The desalted sample is subjected to a mass spectrometer such as MS / MS to determine its primary structure. In this case, quantitative analysis is possible by labeling the fragments to be measured in advance. The labeling may be performed when the second protein is fragmented or after separation (elution) from the first protein. The labeling substance used for labeling may be any labeling substance known in the art such as a fluorescent substance or a radioactive substance. In addition, when only the quantification is intended, the labeled fragment is simply subjected to mass spectrometry, and the primary structure analysis need not be performed.

  The present invention is also a system for identifying a binding site between interacting proteins, comprising means for storing a first protein immobilized on a support, and means for fragmenting a second protein. There is also provided a system having means for capturing the second protein fragment by the first protein and eluting, and means for analyzing the amino acid sequence of the eluted second protein fragment by MS / MS.

  By the method and system of the present invention, an allergic test for identifying a specific allergen or epitope site to which an antibody in a subject reacts can be performed. Alternatively, in an autoimmune disease (rheumatoid arthritis, collagen disease, primary biliary cirrhosis, autoimmune diabetes, thyroiditis, etc.), an autoantigen that causes autoantibody generation that is the cause of the disease can be detected and identified. Since the antigen recognizing ability of autoantibodies changes as the disease progresses, and these changes often appear as a precursor to the onset, this system is expected to be particularly effective for early diagnosis of the disease. In addition, using the method or system of the present invention, a drug screening that identifies an epitope sequence that specifically binds to a target protein such as an antibody or a target receptor, and performs drug design or search for candidate substances based on the sequence. Can also be done. Furthermore, by clarifying differences in these epitope sequences based on disease stages and individual differences, it becomes possible to perform custom-made medication and treatment suitable for each patient.

  According to the present invention, in a protein-protein interaction including an antigen-antibody reaction, the binding site can be easily and efficiently identified (primary structure determination). The method of the present invention can quantitatively analyze a very small amount of sample (about several μl) and can simultaneously analyze two or more kinds of proteins that interact with one protein.

  Many allergies that develop after adults are thought to depend on lifestyle habits in childhood.Therefore, if allergens are identified during early childhood and allergens are identified and attention is paid to lifestyle habits such as eating early, It becomes possible to prevent the onset of allergies. However, since it is difficult to collect a large amount of specimens in the examination of infants and infants, the method of the present invention that can be identified by a trace quantity specimen is a very effective means. Furthermore, the present invention enables identification of a binding site quickly and easily even in protein-protein interactions other than antigen-antibody reactions. Therefore, according to the present invention, development of a medicine with high effect and few side effects and custom-made medical care are promoted.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

[Example 1]
1 to 3 show a process for identifying an interprotein binding site using a protein microarray. Here, (1) immobilization of protein A to be analyzed on a support, (2) fragmentation of protein B to be analyzed, (3) capture of protein B fragment by immobilized protein A, (4 Although the experimental operation is proceeding in the order of: elution of the captured protein B fragment and (5) desalting and MS / MS measurement, the present invention is not necessarily limited to the method shown here.

  In this embodiment, a spot seal 302, a micro pipettor 303, a sealed container 306, a gas phase incubator 307, and a cleaning container 308 for spotting a sample or a reagent on a support are used.

  The spot seal 302 is a hydrophobic sheet of 25 mm length x 55 mm width in which spot holes having a diameter of 2 mm are arranged at intervals of 5 mm in 4 rows and 4.5 mm intervals in 12 rows. The back surface is tacky, and the sample and the reagent can be spotted at the same position on the support in the same size by pasting on the support in advance. The micro pipetter 303 is for spotting a sample or a reagent 304 in an arbitrary amount on a support. The sealed container 306 is for keeping the humidity inside the container for a long time by sealing the support together with a small amount of water droplets 305 and preventing evaporation of samples and reagents on the support. The gas phase incubator 307 is for maintaining a temperature suitable for the binding reaction of the sample and the reagent. The washing container 308 is used for the purpose of removing molecules that are not bound to the support by shaking the support together with the washing solution.

  (1) In immobilizing protein A to be analyzed on a support, a spot seal 302 is attached to the support 301, and protein A to be analyzed is manually dropped using a micropipette 303. The support is placed in the sealed container 306 together with the water droplet 305, and is kept warm in the incubator 307 to perform the binding reaction of protein A onto the support. Unbound protein A is removed by washing.

  (2) In fragmentation of protein B to be analyzed, protein B to be analyzed is fragmented by chemical or enzymatic treatment.

  (3) In capturing the protein B fragment by the immobilized protein A, the protein B sample fragmented in (2) is added to the protein A immobilized on the support in (1) and incubated for a certain period of time. Only peptide fragments having a sequence corresponding to the recognition site are captured by protein A, and the other fragments are removed by a washing operation.

  (4) For elution of the captured protein B fragment, an elution reagent is added to the protein-immobilized spot, and after incubation for a certain period of time, the peptide fragment eluted from the support surface into the elution reagent is recovered.

  (5) In desalting and MS / MS measurement, the salt in the elution reagent is removed using a commercially available desalting device (Millipore's Zip-Tip or the like) 312, and then measured by the mass spectrometer 313. The peptide peak obtained by MS analysis is analyzed for its amino acid sequence by MS / MS analysis.

[Example 2]
In this example, an anti- (His) ₆ antibody that recognizes a polypeptide chain consisting of 6 histidines is immobilized on a support, and a protease digest of an antigen retaining the (His) ₆ sequence is added to the (His) ₆ An experiment in which the sequence site is captured by an antibody and then eluted from the support and sequenced by MS / MS measurement is described.

1. Immobilization of antibody on support In this example, ProteoChip (Type A, Proteogen Inc.) was used as a support. ProteoChip is a protein chip in which a protein binding reagent 'ProLinker', a kind of calix crown derivative, is coated on a slide glass (length 26 mm x width 76 mm), and the protein is immobilized on the surface by interaction with ProLinker. Can do.

On the surface of the ProteoChip, a spot seal 302 (disclosed in the design No. 1213441) 302 for spotting a sample or a reagent at the same position at the same size was attached in advance. Anti- (His) ₆ antibody (MAB050, R & D Systems Inc.) is diluted to 100 μg / ml using PBS (pH 7.4) containing 30% Glycerol, and is supported against support 301 using micropipette 303. 1.5 μl was added dropwise.

The support was placed in a sealed container 306 with a water drop 305 at the corner and incubated overnight at 37 ° C. to immobilize the anti- (His) ₆ antibody on the support. After immobilization, the support was immersed in 50 ml of washing solution (PBS containing 0.5% Tween 20, pH 7.8) and shaken for 10 minutes to remove unbound antibody. After washing, excess water was removed with filter paper.

2. Fragmentation of antigen to be analyzed by protease
(His) ₆ fusion recombination model protein (obtained from the Tohoku University Institute of Multidisciplinary Research for Multidisciplinary Materials) obtained 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl and 2.5 mM CaCl ₂ After dilution to 0.5 mg / ml, 30 U thrombin (27-0846-01, Amercham Bioscience) was added and incubated overnight at room temperature. To confirm digestion, a part of the digest was collected and Ni-NTA carrier (30210, QIAGEN) was added. With the index that the dye retained by the model protein as a cofactor does not bind to the carrier, the (His) ₆ sequence was confirmed to be detached.

3. Capture of antigen fragments by immobilized antibody 1.5 μl of thrombin digested (His) ₆ fusion recombinant model protein was added dropwise to the support using a micropipetter 303. The support was placed in a sealed container 306 with a water drop 305 in the corner, and incubated overnight at 37 ° C., and the peptide carrying the (His) ₆ sequence was supplemented with anti- (His) ₆ antibody. The support was immersed in 50 ml of washing solution (PBS containing 0.5% Tween 20, pH 7.8) and shaken for 10 minutes to remove unbound peptide. After washing, excess water was removed with filter paper.

4). Elution of the captured antigen fragment 1.5 μl of 6M guanidine hydrochloride as an elution reagent 310 was dropped onto the support using a micropipette 303. The support was placed in a sealed container 306 with a water drop 305 at the corner and incubated at room temperature for 10 minutes to dissociate the peptide bound to the anti- (His) ₆ antibody. 6M guanidine hydrochloride was collected using a micropipetter. Again 1.5 μl of 6M guanidine hydrochloride was added to the support and the same recovery procedure was repeated.

5). Desalting and MS / MS Measurement Desalting was performed using a commercially available desalting device Zip-Tip C ₁₈ (ZTC1 8S0 08, Millipore) 312. The desalted sample was subjected to centrifugal concentration to remove the organic solvent (acetonitrile) used during desalting. The sample after desalting was measured with a liquid chromatograph mass spectrometer NanoFrontier (Hitachi High Technologies) 313. The sequence of the detected peptide ion was determined by MS / MS measurement.

6). Experimental Results FIG. 4 shows the results of mass spectrometry in which MS / MS measurement was performed. The horizontal axis is the m / z ratio, and the vertical axis is the ionic strength. As a result, a peptide “GSSHHHHHHSSGLVPR (SEQ ID NO: 1)” was identified. This completely matched the amino acid sequence of the peptide containing the (His) ₆ sequence predicted from the nucleotide sequence.

  The present invention is useful in the medical field including drug discovery, treatment and diagnosis. In particular, by identifying the binding site between allergens and IgE in the body, it will be possible to develop allergy treatments that are more effective and have fewer side effects. In addition, by analyzing the allergen (epitope site) unique to the patient in the diagnosis, treatment suitable for the individual constitution is possible.

FIG. 1 shows the experimental flow for protein binding site identification. FIG. 2 shows the principle of protein binding site identification. FIG. 3 shows an experimental procedure using a protein microarray. FIG. 4 shows the results of MS / MS measurement (horizontal axis: m / z ratio, vertical axis: ionic strength).

Explanation of symbols

201: Protein A
202: Protein B
203: Fragmented protein B
204: Protein elution reagent
301: Support
302: Spot seal
303: Micro Pipetter
304: Protein A
305: Water drops
306: Sealed container
307: Incubator
308: Cleaning container
309: Fragmented protein B
310: Protein elution reagent
311: Eluted protein B fragment
312: Desalination device
313: Mass spectrometer

SEQ ID NO: 1 Fragment bound to anti- (His) ₆ antibody

Claims (16)

  A method for identifying a binding site between interacting proteins, wherein the first protein is immobilized on a support, the second protein is fragmented, added to the first protein, and the first protein The second protein fragment captured by the second protein fragment is separated, the amino acid sequence of the separated second protein fragment is analyzed by mass spectrometry, and the binding site of the second protein to the first protein is determined. A method characterized by identifying.   2. The method of claim 1, wherein the first protein is a receptor and the second protein is its ligand.   2. The method of claim 1, wherein the first protein is an antibody and the second protein is an antigen.   4. The method of claim 3, wherein the antigen is an allergen.   The method according to claim 1, wherein the first protein is immobilized on a support via a linker.   The method according to claim 1, wherein the fragmentation of the second protein is performed by an enzyme treatment.   The method according to claim 1, wherein the mass spectrometry is performed using MS / MS.   2. The method of claim 1, wherein the second protein fragment is labeled.   The method according to claim 1, wherein two or more proteins are used as the second protein.   A system for identifying a binding site between interacting proteins, comprising: a means for storing a first protein immobilized on a support; a means for fragmenting a second protein; A system comprising: means for capturing and eluting the second protein fragment by protein; and means for analyzing the amino acid sequence of the eluted second protein fragment by MS / MS.   11. The system of claim 10, wherein the first protein is a receptor and the second protein is its ligand.   11. The system according to claim 10, wherein the first protein is an antibody and the second protein is an antigen.   The system of claim 12, wherein the antigen is an allergen.   The system according to claim 10, further comprising means for labeling the second protein fragment.   An allergy test using the method according to claim 1.   Drug screening using the method according to claim 1.

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