JP2004283114A - Method for exhaustively analyzing protein kinase activity by using surface plasmon resonance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To establish a method capable of exhaustively profiling kinetics in various protein kinase by a rapid and simple technique not requiring a specific technique. <P>SOLUTION: An array is obtained by immobilizing at least one kind of corresponding peptide which becomes a substrate for at least one kind of proteinase onto a metal thin film of the substrate. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】本発明において用いられる表面プラズモン共鳴イメージング装置の一例を示す図である。
【図２】実施例１において、アレイ上に２種類の基質ペプチドを固定化したパターンを示す図である。
【図３】実施例２において、ＳＰＲ解析を行った結果である。
【図４】実施例２において、ＳＰＲイメージングを行った結果である。
【図５】実施例４において、オートラジオグラフィを行った結果である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for analyzing protein kinase activity using an array in which one or more kinds of peptides recognizing protein kinase are immobilized on a metal substrate. Particularly, by using an analysis technique based on surface plasmon resonance (hereinafter sometimes referred to as SPR), rapid, efficient and simple analysis of protein kinase activity can be realized. More specifically, a novel method for analyzing protein kinase activity using surface plasmon resonance imaging (hereinafter sometimes referred to as SPR imaging) that monitors the state of interaction between substances analyzed by SPR. It is.
[0002]
[Prior art]
In recent years, research on intracellular signal transduction has progressed dramatically, not only how signals are transmitted to nuclei from cell surface receptors activated by growth factors and cytokines, but also cell cycle, adhesion, The actual state of various signal transduction pathways controlling movement, polarity, morphogenesis, differentiation, life and death, etc. has been revealed. These signaling pathways do not function independently, but cross-talk with each other and function as a system. The causes of various diseases including cancer have been described as abnormalities in these signal transduction pathways.
[0003]
It is known that various types of protein kinases play an important role in the above-mentioned signal transduction pathways in a complicatedly related manner. If we could comprehensively analyze the activity of these protein kinases and profile their intracellular dynamics at once, we would not only be able to perform basic research in cell biology and pharmacy, but also in fields such as drug development and clinical application. It is expected to make a significant contribution. However, a technique that can simultaneously and easily profile the kinetics of various protein kinases has not yet been established so far.
[0004]
As a related technique already reported, for example, it has been described that the activity of c-Src kinase, which is a kind of tyrosine kinase, was evaluated using a peptide array (for example, see Non-Patent Document 1). In addition, for p60 tyrosine kinase, protein kinase A (hereinafter also referred to as PKA), etc., a phosphorylation detection system using a fluorescently labeled antibody using an array in which each substrate peptide is immobilized on a glass slide. (For example, see Non-Patent Documents 2 and 3) or a radioactive substance ([γ ³² There is an example (for example, see Non-Patent Documents 4, 5, and 6) which describes a detection system for a kinase reaction on an array using [P] ATP). However, none of the prior arts discloses a sufficient technique as a method for simultaneously and efficiently profiling the kinetics of various protein kinases. Further, in any of the above methods, it is necessary to use a fluorescent substance or a radioactive substance, and there are significant problems in that analysis requires time and effort, handling is difficult, and special techniques and facilities are required.
[0005]
[Non-patent document 1]
Benjamin T. Houseman et al., Nature Biotechnology, Volume 20, pages 270-274 (issued March 2002).
[0006]
[Non-patent document 2]
Bioorganic & Medical Chemistry Letters, Vol. 12, pp. 2085-2088 (published in 2002)
[0007]
[Non-Patent Document 3]
Bioorganic & Medical Chemistry Letters, Vol. 12, pp. 2079-2083 (published in 2002)
[0008]
[Non-patent document 4]
Current Opinion in Biotechnology Volume 13, pp. 315-320 (issued in 2002)
[0009]
[Non-Patent Document 5]
The Journal of Biological Chemistry Volume 277, 27839-27849 (published in 2002)
[0010]
[Non-Patent Document 6]
Science Vol. 289, pp. 1760-1763 (2000)
[0011]
[Problems to be solved by the invention]
The present invention seeks to establish a method capable of comprehensively profiling the kinetics of various protein kinases by a simple method that is quick and does not require special techniques.
[0012]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in view of the above circumstances, and as a result, using an array in which a peptide recognizing protein kinase is immobilized on a metal-deposited substrate, particularly the mutual interaction between substances on the array. The present inventors have found that detecting the action by the SPR imaging method is extremely useful for rapid analysis of various protein kinase kinetics, and arrived at the present invention.
[0013]
That is, the present invention has the following configuration.
Item 1. An array in which at least one corresponding peptide serving as a substrate for at least one protein kinase is immobilized on a metal thin film on a substrate.
Item 2. The peptide is a cGMP-dependent protein kinase family, a cAMP-dependent protein kinase (PKA) family, a myosin light chain kinase family, a protein kinase C (PKC) family, a protein kinase D (PKD) family, a protein kinase B (PKB) family; Item 2. The array according to Item 1, which is a substrate of at least one protein kinase selected from the group consisting of a protein kinase family belonging to a MAP kinase (MAPK) cascade, a Src tyrosine kinase family, and a receptor tyrosine kinase family.
Item 3. Item 2. The array according to Item 1, wherein at least two types of peptides, each of which is a separate protein kinase substrate, are immobilized on a metal thin film.
Item 4. Item 5. Analysis of protein kinase activity, wherein a sample containing protein kinase and a nucleoside triphosphate are allowed to act on the peptide of the array according to any one of Items 1 to 3, and the phosphorylated peptide is detected. Method.
Item 5. Item 3. The method for analyzing protein kinase activity according to Item 2, wherein the phosphorylated peptide is detected by utilizing surface plasmon resonance.
Item 6. Item 6. The method according to Item 5, wherein the phosphorylated peptide is detected by surface plasmon resonance imaging.
Item 7. Item 7. The method according to any one of Items 4 to 6, wherein the phosphorylated peptide is reacted with an antibody that recognizes a phosphorylation site in the peptide to form a complex, and the complex is detected.
Item 8. Item 8. The method according to Item 7, wherein the phosphorylation site recognized by the antibody is a tyrosine residue.
Item 9. Item 5. The method for analyzing protein kinase activity according to Item 4, wherein the phosphorylation site recognized by the antibody is a serine residue or a threonine residue.
Item 10. Item 10. The method according to any one of Items 4 to 9, wherein the peptide has a cysteine residue at one or both of the N-terminus and the C-terminus.
Item 11. Item 4. The array according to any one of Items 1 to 3, for performing protein kinase activity analysis using surface plasmon resonance.
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described specifically.
[0015]
The method for detecting a phosphorylated peptide on a substrate in the present invention can be performed using a labeling compound such as a radioactive substance, a fluorescent substance, and a chemiluminescent substance which are well known in the art. However, it is more preferable to apply an optical detection method such as surface plasmon resonance (SPR), ellipsometry (hereinafter, referred to as ellipsometry), and sum frequency generation (hereinafter, referred to as SFG) spectrometry. Above all, SPR is particularly preferable because it is not necessary to obtain a phase difference, and it is possible to obtain a change in film thickness on the order of nm on the surface only by obtaining reflected light intensity. The SPR imaging method is more preferable because it can observe a wide range and can observe substance interaction in an array format.
[0016]
In one preferred embodiment of the present invention, the phosphorylated peptide is detected by utilizing a substance interaction between a substance capable of recognizing the phosphorylated peptide and the phosphorylated peptide. Preferred examples of the substance capable of recognizing a phosphorylated peptide include an antibody.
[0017]
Phosphorylation of the peptide can be performed by applying a test sample that may have protein kinase and a nucleoside triphosphate, for example, ATP, onto the array of the present invention. For example, a test sample which may have protein kinase in a buffer and a nucleoside triphosphate are added, and at a temperature of about 10 to 40 ° C, preferably at a temperature of 30 to 40 ° C, for about 10 minutes to 6 hours, preferably The peptide can be phosphorylated by reacting for about 30 to 1 hour. If necessary, cAMP, cGMP, Mg ²⁺ , Ca ²⁺ For example, a substance that assists phosphorylation may be allowed to coexist.
[0018]
Kinase profiling protein kinases include enzymes that phosphorylate the side chains of amino acids such as tyrosine, serine, threonine, and histidine of proteins. For example, cGMP-dependent protein kinase family, cAMP-dependent protein kinase ( PKA) family, myosin light chain kinase family, protein kinase C (PKC) family, protein kinase D (PKD) family, protein kinase B (PKB) family, protein kinase family belonging to MAP kinase (MAPK) cascade, Src tyrosine kinase family And the receptor tyrosine kinase family.
[0019]
In the present invention, the purpose of a peptide is to comprehensively profile the kinetics of protein kinase, so that one kind of peptide is phosphorylated only by one kind of protein kinase, and is not phosphorylated by another kind of protein kinase. preferable. For example, the PKA substrate is exemplified by the peptide of SEQ ID NO: 1, and the MAPK substrate is exemplified by the peptide of SEQ ID NO: 2. Peptide sequences serving as substrates for protein kinases including PKA and MAPK are known or can be appropriately selected based on known sequences. If the array of the present invention has immobilized peptides of a type corresponding to a plurality of types of protein kinases whose dynamics need to be grasped, profiling of all protein kinases can be performed with one array, which is preferable. Of course, a peptide corresponding to only one type of protein kinase may be immobilized on one array, and protein kinase profiling may be performed using a required number of arrays.
[0020]
Ellipsometry irradiates a sample with light, and can measure a film thickness and a refractive index from a change in a deflection state caused by interference between light reflected on the front surface of the thin film and light reflected on the back surface of the thin film. That is, it is a means for evaluating the ratio of the absolute value of the reflectance and the ratio of the phase change to the p-polarized light and the s-polarized light. Above all, spectroscopic ellipsometry, in which ellipsometry is measured while changing the wavelength, is preferable because a change in film thickness on the surface can be detected very sensitively.
[0021]
SFG is a kind of second-order nonlinear optical effect, and is a phenomenon in which two types of incident light (frequency ω1 and frequency ω2) having different frequencies are mixed in a medium to generate light of ω1 + ω2 or ω1-ω2. In particular, when visible light is used as ω1 and variable wavelength infrared light is used as ω2, vibrational spectroscopy similar to infrared spectroscopy can be performed. Since this method has good surface selectivity, vibration spectroscopy of molecules at the monomolecular film level is possible, and is useful as a very sensitive surface analysis method.
[0022]
As described above, in one particularly preferred embodiment, it is particularly preferred that the present invention comprehensively analyze various protein kinase activities using SPR. In the SPR, an evanescent wave is generated by a polarized light beam irradiated on a metal, and oozes on the surface, excites surface plasmons, which are surface waves, and consumes light energy to reduce the intensity of reflected light. The resonance angle at which the intensity of the reflected light is significantly reduced depends on the thickness of the layer formed on the surface of the metal. Therefore, the substance or substance aggregate to be examined is immobilized on the metal surface, and the interaction with the substance or substance aggregate in the sample is detected by the change in the resonance angle or the change in the reflected light intensity at a certain angle. It is possible. Therefore, SPR does not require a label and is useful as a quantitative method capable of real-time evaluation.
[0023]
The SPR imaging method using this SPR is a method of irradiating a polarized light beam over a wide area and analyzing the reflected image to monitor the state of interaction between substances by making full use of image processing technology and the like. In addition, it is possible to screen a chip on which a plurality of substances are immobilized, and to observe the morphology of an object adsorbed on the surface with high sensitivity.
[0024]
In the SPR imaging method, a means for irradiating a chip with a polarized light beam over a wide area in order to analyze a reflected image and ensuring sufficient illuminance of the light beam is required. FIG. 1 shows an example. It is more preferable that the illuminance of the polarized light beam is higher because the sensitivity of the sensor increases.
[0025]
Although the type of the light source is not particularly limited, it is preferable to use light including near-infrared light whose change in the SPR resonance angle is particularly sensitive. Specifically, a white light source that can irradiate light over a wide range, such as a metal halide lamp, a mercury lamp, a xenon lamp, a halogen lamp, a fluorescent lamp, and an incandescent lamp, can be used. A halogen lamp which is sufficiently high, has a simple light power supply device and is inexpensive is particularly preferable.
[0026]
An ordinary white light source has a drawback in that light and darkness unevenness occurs in a filament portion. When the light from the light source is irradiated as it is, unevenness in brightness and darkness occurs in an image obtained by reflection, making it difficult to evaluate screening and morphological changes. Therefore, as a means for uniformly irradiating the chip with light, a method of passing light through a pinhole and then converting the light into parallel light is preferable.
[0027]
The means for passing through the pinhole is preferable as a means for obtaining a light beam having a uniform brightness, but there is a disadvantage that the illuminance is reduced if the light passes through the pinhole as it is. Therefore, as a means for securing sufficient illuminance, it is preferable to use a method in which a convex lens is provided between the pinhole and the light source, and light is collected and passed through the pinhole.
[0028]
Since the white light source is emitted light, it is necessary to use a convex lens to convert the white light into parallel light before focusing. By installing a light source near the focal length of the convex lens, parallel light can be obtained. By installing another convex lens and installing a pinhole near the focal length of the lens, it is possible to pass the condensed light through the pinhole. The light that crosses and passes through the pinhole is converted into parallel light by a CCTV lens for a camera, and the cross-sectional area of the parallel light flux obtained at that time is 10 to 1000 mm. ² It is preferred to adjust to. This method enables a wide range of screening and morphology observation.
[0029]
When monitoring the interaction, the polarized light beam is irradiated on the opposite surface of the metal thin film on which the substance or the aggregate of the substance is immobilized. The polarized light beam is applied to the opposite surface of the metal thin film on which the substance or the aggregate of the substance is immobilized, and the reflected light beam is obtained. The light beam reflected from the metal thin film passes through a near-infrared wavelength light interference filter, passes only light near a certain wavelength, and is photographed by a CCD camera.
[0030]
The center wavelength of the optical interference filter is preferably from 600 to 1000 nm, at which the sensitivity of SPR is high. The wavelength width of the wavelength at which the transmittance of the optical interference filter is half of the maximum value is called the half-value width. The smaller the half-value width is, the sharper the wavelength distribution becomes, and more specifically, the more preferable is the half-value width of 100 nm or less.
[0031]
An image captured by a CCD camera through an optical interference filter is captured by a computer, and a change in brightness of a certain portion can be evaluated in real time, and an entire image can be evaluated by image processing. In this way, it is possible to screen a chip on which a plurality of substances are immobilized, and to observe the morphology of an object adsorbed on the surface with high sensitivity.
[0032]
The SPR chip used in the present invention is preferably formed of a metal substrate having a metal thin film formed on a transparent substrate, and directly or indirectly, chemically or physically, forming a substance or substance on the metal thin film. A slide on which the aggregate is fixed is used. The material of the substrate is not particularly limited, but it is preferable to use a transparent material. Specific examples include glass and plastics such as polyethylene terephthalate (PET), polycarbonate, and acrylic. Among them, glass is particularly preferred.
[0033]
The thickness of the substrate is preferably about 0.1 to 20 mm, more preferably about 1 to 2 mm. In order to achieve the purpose of evaluating the reflection image from the metal thin film, the SPR resonance angle should be as small as possible, so that the captured image does not have a risk of shaking and the analysis is easy. Therefore, the refractive index n of the transparent substrate or the transparent substrate and the prism in contact therewith _D Is preferably 1.5 or more.
[0034]
Examples of the metal constituting the metal thin film include gold, silver, copper, aluminum, and platinum. These may be used alone or in combination, but among them gold is particularly preferable. The method for forming the metal thin film is not particularly limited, but known methods include, for example, an evaporation method, a sputtering method, and an ion coating method. Above all, a method by vapor deposition is preferable. Further, the thickness of the metal thin film is preferably about 10 to 3000 °, more preferably about 100 to 600 °.
[0035]
One particularly preferred embodiment of the present invention uses an array in which at least one, preferably a plurality of peptides serving as protein kinase substrates are immobilized on a metal-deposited substrate, and It is characterized in that a kinase-containing solution such as a cell lysate is allowed to act thereon, and the state of their interaction is detected by SPR or SPR imaging. In the present invention, a peptide serving as a substrate for a protein kinase refers to a peptide having a property of undergoing a phosphorylation reaction by the protein kinase.
[0036]
The length of the peptide is not particularly limited, but generally a peptide having 100 amino acid residues or less is used. Preferably, those having about 5 to 60 amino acid residues, more preferably about 10 to 25 amino acid residues are used. The peptide may be a peptide obtained by chemical synthesis based on a known technique, or a peptide produced by a genetic engineering technique may be used. In order to facilitate desorption to the substrate, biotin or a cysteine residue having a thiol group is added to one end of the peptide, or oligohistidine (His-tag), glutathione-S-transferase It is also useful to use a tag to which a commonly used tag such as (GST) is added.
[0037]
The method of immobilizing the peptide on the metal thin film is not particularly limited, but it is more preferable to preliminarily introduce a functional group that can be easily immobilized on the surface of the metal thin film and then immobilize the substrate peptide. preferable. Examples of the functional group include an amino group, a mercapto group, a carboxyl group, and an aldehyde group. In order to introduce these functional groups into the surface of the metal thin film, it is preferable to use a generally used alkanethiol derivative.
[0038]
At that time, J.I. M. Brockman et al. Am. Chem. Soc. Vol. 121, pp. 8044-8051 (1999), a method of immobilizing via an alkanethiol layer and modifying the background with PEG (polyethylene glycol) can also be used. Good. Further, in order to further suppress non-specific effects, immobilizing the peptide after binding a derivative having the above-described functional group introduced to the terminal of PEG to alkanethiol also has a spacer effect. Useful in
[0039]
Specifically, for example, a metal thin film is bonded to a water-soluble polymer such as carboxymethyl dextran or PEG whose terminal is modified with a carboxyl group to introduce a carboxyl group to the surface, and EDC (1-ethyl-3, As a ester of NHS (N-hydroxysuccinimide) using a water-soluble carbodiimide such as 4-dimethylaminopropylcarbodiimide), a method of reacting an activated carboxyl group with an amino group of a peptide or protein can be mentioned. Alternatively, after the surface is modified with maleimide, it may be immobilized via an amino acid residue containing a thiol group such as cysteine. In this case, the cysteine residue is preferably added to one end of the peptide. In order to reduce non-specific effects, the latter method of immobilization via a thiol group is more preferable, but is not particularly limited.
[0040]
The method of immobilizing a tagged peptide such as His-tag or GST described above is also very simple and useful. In this case, after introducing an amino group or a carboxyl group into the metal surface via the alkanethiol as described above, it is preferable to introduce NTA (nitrilotriacetic acid) and glutathione onto the metal thin film, respectively. In the case of His-tag, the array in which NTA is introduced is treated with nickel chloride, and then the substrate is immobilized.
[0041]
In the present invention, in order to monitor the phosphorylation of the substrate on the array with high sensitivity, it is more preferable to perform analysis using an antibody that recognizes a phosphorylation site in the peptide. As the antibody, for example, an antibody that recognizes a phosphorylated tyrosine residue or a phosphorylated serine residue or threonine residue is preferably used. The antibody may be either a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is more preferable in terms of detection specificity and sensitivity. The antibody class may be any of IgG, IgM and the like. It is preferable to use a sufficiently purified antibody. For example, ascites of a mouse or a culture supernatant of a hybridoma cell containing a monoclonal antibody can be used as it is. Examples of the protein kinase include various tyrosine kinases or serine / threonine kinases. The type of these protein kinases is not particularly limited, and can be basically applied to all types of protein kinases.
[0042]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not particularly limited to the examples.
Example 1: Preparation of peptide array
First, a gold-deposited glass plate (LAK-10 manufactured by Osaka Vacuum Industry Co., Ltd .; size: 18 mm × 18 mm × 1 mm, coating content: first layer of chromium 20Å, second layer of gold 450Å; refractive index 1.72) The sample was immersed in a 20 mM ethanol solution of SUNBRIGHT MASH-50H (manufactured by NOF Corporation) at a concentration of 1 mM for 2 hours. This glass plate was washed with ethanol and water in that order, dried, and then fixed with a photomask placed on the glass plate, and irradiated with ultraviolet light (using an Optical Module SX-UI500H manufactured by Ushio Inc .; 500 watts) for 2 hours. went. The photomask has 49 square holes each having a side of 500 μm (formed of 7 × 7 patterns), and the pitch between the centers of the holes is designed to be 1 mm. After the irradiation with ultraviolet rays, the glass plate was washed with ethanol, and further immersed in 20 ml of an ethanol solution of 1 mM MOAM (8-amino-1-octanethiol, hydrochloride; Dojindo Chemical) for 1.5 hours to remove amino groups on the array surface. Introduced.
[0043]
The solution was dissolved in a 10 mg / ml NHS-PEG-MAL (molecular weight: 3400; manufactured by Shearwater) solution (pH 7.4, 10 mM phosphate buffer (containing 100 mM NaCl)) on the glass plate treated as described above. ) 200 μl was placed and allowed to stand at room temperature for 2 hours. The glass plate was washed with ethanol and water in that order, dried, and then spotted on the photo pattern formed on the glass plate in an amount of 10 nl. The peptide solution (dissolved in a 10 mM phosphate buffer (containing 100 mM NaCl) at pH 7.4) used for the spot had a concentration of 100 μg / ml. For spotting, DNA-GR manufactured by Nichiryo was used. As the substrate peptide, a synthetic peptide having the amino acid sequence shown in SEQ ID NO: 1 (which serves as a substrate for PKA) and SEQ ID NO: 2 (which serves as a substrate for MAPK) was used. The immobilization pattern of each substrate was as shown in FIG. The glass plate on which the peptide solution was spotted was allowed to react at room temperature overnight in a wet environment at room temperature. Thus, an array having the kinase substrate peptide immobilized thereon was prepared.
Example 2: SPR analysis
The peptide array prepared in Example 1 was washed with water and ethanol, dried and then set on an SPR device (SPRIimager manufactured by GWC Instruments Inc.) for analysis. As a running buffer, a 50 mM Tris-HCl buffer (pH 7.4, containing 50 mM magnesium chloride and 1 mM ATP (adenosine triphosphate)) was used. A running buffer was supplied using a plunger pump (Model-021 manufactured by From). The temperature was set at 30 ° C. In the phosphorylation reaction of the substrate, a commercially available cAMP-Dependent Protein Kinase Catalytic Subunit (promega) was used as PKA. This was performed for 30 minutes while circulating a PKA solution (about 200 U / ml) diluted 400-fold with the running buffer. Then, ab6639 (manufactured by abcam) or PSR-45 (manufactured by Sigma), which is an antibody that recognizes a phosphorylated serine residue, was allowed to act thereon. The antibody solution used was diluted 1000-fold with the above running buffer.
[0044]
The result obtained as described above is shown in FIG. A specific SPR signal was confirmed only in the spot of the PKA substrate, and almost no signal was confirmed in the spot of the MAPK substrate.
[0045]
The same array as described above was set in an SPR apparatus in the same manner as above, and the same examination was performed for the phosphorylation reaction by MAPK (mitogen-activated protein kinase). As MAPK, MAP Kinase, Activated, Rat, Recombinant (manufactured by Calbiochem) was used (diluted 400-fold), and the same procedure was performed except that the reaction time was 90 minutes. As a result, contrary to the case of PKA, a specific SPR signal was confirmed only in the spot of the MAPK substrate, and almost no signal was confirmed in the spot of the PKA substrate.
[0046]
FIG. 4 shows the result of performing SPR imaging. At the time of SPR analysis in Example 2, an image was captured by a CCD camera every 5 seconds, and from an image captured at a time after the antibody reaction, an image at a time before the reaction was converted to image operation software Scion Image ( Scion Corp.). When PKA (FIG. 4-A) and MAPK (FIG. 4-B) were allowed to act, spots were confirmed only on the immobilized portions of each substrate.
Example 3: Preparation of array for autoradiography
First, the same gold-deposited glass plate as used in Example 1 was immersed in 20 ml of a 1 mM MOAM ethanol solution for 1.5 hours to introduce amino groups on the array surface. After washing and drying in the order of ethanol and water, 500 μl of a 4 mM mPEG-SPA (molecular weight: 2,000; manufactured by Shearwater Cop.) Solution was placed on a slide, and allowed to stand for 1.5 hours. Further, after washing and drying in the order of ethanol and water, a photomask was placed and fixed on a glass plate in the same manner as in Example 1, and ultraviolet irradiation was performed for 2 hours. A photomask having 16 square holes each having a side length of 500 μm (a pattern of 4 × 4 patterns) was used. After the ultraviolet irradiation, the glass plate was washed with ethanol and immersed in a 20 mM ethanol solution of 1 mM MHA (7-carboxy-1-heptanethiol; Dojindo Chemical) for 1.5 hours to form a carboxyl group surface.
[0047]
The array was washed with ethanol and water in that order, dried, and slided with 200 μl of 0.2 M EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) /0.05 M NHS (N-hydroxysuccinimide) solution. Placed on top and left for 1 hour to activate the surface. After washing and drying the array in the order of ethanol and water, 0.1 μl of the PKA substrate and 0.1 μl of the MAPK substrate were spotted in a pattern as shown in FIG. The immobilization reaction was performed overnight at room temperature to prepare an array immobilized via the amino groups of the peptide.
Example 4: Detection of phosphorylation by autoradiography
On the array prepared in Example 3, γ ³² 200 μl of a PKA solution containing P-ATP was dropped and reacted at 30 ° C. for 30 minutes. The same PKA as used in Example 2 was used and diluted 400-fold with a 50 mM Tris-HCl buffer (containing 50 mM magnesium chloride) for use in the reaction. Also, [γ ³² [P] ATP (manufactured by Amersham Bioscience) was allowed to coexist at about 5 Ci / mmol. After the reaction, the array was washed three times with water, air-dried, and subjected to autoradiography. The RI analysis used BAS-1800II (manufactured by Fuji Photo Film). Exposure to the imaging plate (IP sheet) was performed for 30 minutes. The results are shown in FIG. A spot was specifically confirmed only at the site where the PKA substrate was immobilized.
[0048]
【The invention's effect】
As described above, according to the method of the present invention, no special technique is required, and particularly when SPR is used, there is no need to use a label such as a fluorescent substance or a radioactive substance, which is very simple and rapid. It has become possible to analyze various protein kinase dynamics. By utilizing the present invention, it is possible to comprehensively analyze various types of protein kinase signals, and to effectively profile protein kinase dynamics in cells associated with introduction of a gene whose function is unknown or drug administration. Can be. This is expected to be applied to genomic drug discovery, such as functional analysis from new genes and approaches to drug discovery.
[0049]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing an example of a surface plasmon resonance imaging apparatus used in the present invention.
FIG. 2 is a view showing a pattern in which two types of substrate peptides are immobilized on an array in Example 1.
FIG. 3 shows the result of performing SPR analysis in Example 2.
FIG. 4 shows the result of performing SPR imaging in Example 2.
FIG. 5 shows the result of performing autoradiography in Example 4.

Claims (11)

An array in which at least one corresponding peptide serving as a substrate for at least one protein kinase is immobilized on a metal thin film on a substrate. The peptide is a cGMP-dependent protein kinase family, a cAMP-dependent protein kinase (PKA) family, a myosin light chain kinase family, a protein kinase C (PKC) family, a protein kinase D (PKD) family, a protein kinase B (PKB) family; The array according to claim 1, which is a substrate of at least one protein kinase selected from the group consisting of a protein kinase family belonging to a MAP kinase (MAPK) cascade, a Src tyrosine kinase family, and a receptor tyrosine kinase family. The array according to claim 1, wherein at least two peptides, each of which is a separate protein kinase substrate, are immobilized on a metal thin film. A test material capable of containing protein kinase and a nucleoside triphosphate are allowed to act on the peptide of the array according to any one of claims 1 to 3, and the phosphorylated peptide is detected, whereby the protein kinase activity is detected. analysis method. The method for analyzing protein kinase activity according to claim 2, wherein the phosphorylated peptide is detected using surface plasmon resonance. The method according to claim 5, wherein the phosphorylated peptide is detected by surface plasmon resonance imaging. The method according to any one of claims 4 to 6, wherein the phosphorylated peptide is reacted with an antibody that recognizes a phosphorylation site in the peptide to form a complex, and the complex is detected. The method according to claim 7, wherein the phosphorylation site recognized by the antibody is a tyrosine residue. The method for analyzing protein kinase activity according to claim 4, wherein the phosphorylation site recognized by the antibody is a serine residue or a threonine residue. The method according to any one of claims 4 to 9, wherein the peptide has a cysteine residue at one or both of the N-terminus and the C-terminus. The array according to any one of claims 1 to 3, for performing protein kinase activity analysis using surface plasmon resonance.

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