JP2006050949A - Method for analyzing protein kinase activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for exhaustively profiling kinetics of several protein kinase by detecting phosphorylation of substrate peptide with a simple technique, using an economical material, rapidly and requiring no specific technology. <P>SOLUTION: The method for detecting phosphorylation of peptide on an array obtained by fixing two or more kinds of peptides on a metallic thin film of the substrate, especially the method for analyzing protein kinase activity by surface plasmon resonance is provided, wherein a peptide containing different two or more amino acid residues is fixed as the phosphorylation part of each immobilized peptide and a polyamine zinc complex modified with biotin is acted in detecting phosphorylation. The compounds listed in formula (I) are used as the polyamine zinc complex. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for analyzing protein kinase activity using an array in which two or more types of peptides that recognize protein kinases are immobilized on a metal substrate, and after performing phosphorylation on the array, biotin modification The present invention relates to a method by detecting phosphorylation by acting a chelated compound. In particular, by using an analysis technique based on surface plasmon resonance (hereinafter sometimes referred to as SPR), it is possible to realize a rapid and efficient analysis of protein kinase activity. More specifically, a novel method for analyzing protein kinase activity using a surface plasmon resonance imaging method (hereinafter also referred to as SPR imaging method) in which the state of interaction between substances analyzed by SPR is monitored. It is.

  In recent years, research on intracellular signal transduction has progressed dramatically, not only how signals are transmitted to the nucleus from cell surface receptors activated by growth factors and cytokines, but also cell cycle, adhesion, The reality of various signaling pathways that control movement, polarity, morphogenesis, differentiation, life and death, etc. has become clear. 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 explained as abnormalities in these signal transduction pathways.

  In the signal transduction pathway described above, it is known that various types of protein kinases play an important role in a complex manner. If we can comprehensively analyze the activity of these protein kinases and profile their dynamics in a single cell, we will 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 contribute greatly. However, at present, a technology that can simultaneously and efficiently profile the dynamics of various protein kinases has not yet been established.

As a related technique already reported, for example, it has been reported that the activity of cSrc kinase, which is a kind of tyrosine kinase, was evaluated using a peptide array (see, for example, Non-Patent Document 1). In addition, with respect to p60 tyrosine kinase and protein kinase A (hereinafter also referred to as PKA), 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 examples of reports on detection systems for kinase reactions on arrays using radioactive substances ([γ ³² P] -ATP) ( For example, see Non-Patent Documents 4, 5 and 6. However, in any of the prior arts, a sufficient technique is not disclosed as a method for efficiently and efficiently profiling the dynamics of various protein kinases. In any of the above methods, it is necessary to use a fluorescent substance or a radioactive substance, and there are major problems in terms of the time required for analysis, the difficulty of handling, the necessity of special techniques and facilities, and the like.

  Although detection systems using antibodies are widely applied, especially antibodies that recognize phosphorylated serine and phosphorylated threonine have been found to have sufficient characteristics in terms of their binding specificity and affinity. Therefore, there is a big problem in realizing accurate measurement. In addition, in the case of antibodies, it is necessary to act differently depending on the type of phosphorylated amino acid, and there are many cases where the dependence on the amino acid sequence in the vicinity of the phosphorylated amino acid is high. It is not so advantageous to use as.

Benjamin T. Houseman et al., Nature Biotechnology Volume 20, 270-274 (issued March 2002) Bioorganic & Medical Chemistry Letters Vol. 12, pp. 2085-2088 (issued in 2002) Bioorganic & Medical Chemistry Letters Vol. 12, pp. 2079-2083 (issued in 2002) Current Opinion in Biotechnology Vol. 13, 315-320 (issued in 2002) The Journal of Biological Chemistry Vol. 277, 27839-27849 (issued in 2002) Science 289, 1760-1763 (2000)

  The present invention provides a method capable of comprehensively profiling the kinetics of various protein kinases in particular by detecting phosphorylation of a substrate peptide by a simple method that uses a cheap substance and does not require special techniques. Is to establish.

  As a result of intensive studies in view of the above circumstances, the present inventors have immobilized two or more types of peptides that recognize protein kinases and different types of phosphorylated amino acids on a metal-deposited substrate. After the phosphorylation by protein kinase using the prepared array, the biotin-modified chelate compound is allowed to act, preferably when streptavidin or avidin is further acted on, particularly the interaction between substances on the array Has been found to be extremely useful for rapid and particularly comprehensive analysis of various protein kinase kinetics, and has reached the present invention.

The present invention has the following configuration.
1. A method for detecting phosphorylation of a peptide on an array in which two or more kinds of peptides are immobilized on a metal thin film on a substrate, wherein the two or more kinds of amino acids differ as phosphorylation sites in each of the immobilized peptides A method for analyzing protein kinase activity, wherein a peptide containing a residue is immobilized, and a polyamine zinc complex modified with biotin is allowed to act upon detection of phosphorylation.
2. 1. Analysis of protein kinase activity characterized by using an array on which two or more kinds of peptides containing amino acid residues in combination of two or more of serine, threonine, and tyrosine are used as sites to be phosphorylated Method.
3. 3. A method for analyzing protein kinase activity according to 1 or 2, wherein a polyamine zinc complex modified with biotin is allowed to act, and then avidin or streptavidin is further allowed to act.
4). 4. The method for analyzing protein kinase activity according to any one of 1 to 3, wherein an antibody that recognizes avidin or streptavidin is further reacted after avidin or streptavidin is allowed to act.
5. A method for detecting phosphorylation of a peptide on 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 of a substrate, which is modified with biotin A method for analyzing protein kinase activity, comprising forming a complex of a polyamine zinc complex with avidin or streptavidin and allowing the complex to act.
6). 5. The method for analyzing protein kinase activity according to 5, wherein an antibody that recognizes avidin or streptavidin is further allowed to act after the complex is allowed to act.
7). The analysis method of protein kinase activity in any one of 1-6 using the binuclear zinc complex which has a polyamine compound as a ligand as a polyamine zinc complex.
8). The analysis method of the protein kinase activity in any one of 1-7 using the compound described in Formula (I) as a polyamine zinc complex.
9. 8. The method for analyzing protein kinase activity according to any one of 1 to 8, wherein the phosphorylated peptide is detected using surface plasmon resonance (SPR).
10. 9. The method for analyzing protein kinase activity according to 9, wherein the phosphorylated peptide is detected by a surface plasmon resonance imaging method.

  According to the method of the present invention, no special technique is required, and in particular, when SPR is used, there is no need to use labels for fluorescent substances, radioactive substances, etc. It became possible to analyze the dynamics. In particular, this is a useful technique for simultaneously measuring assays relating to substrates having different types of phosphorylated amino acids. The present invention has a great advantage over the conventional method in that it uses a chelate compound, is inexpensive and easy to handle, and is not affected by the type of phosphorylated amino acid or the amino acid sequence in the vicinity thereof. . By using the present invention, it is possible to comprehensively analyze various types of protein kinase signals, and to effectively profile protein kinase dynamics in cells accompanying gene introduction or drug administration with unknown functions. be able to. This is expected to expand into genomic drug discovery, such as functional analysis from new genes and approaches to drug discovery.

  As a method for detecting a phosphorylated peptide on a substrate in the present invention, it is possible to use a labeling compound such as a radioactive substance, a fluorescent substance, a chemiluminescent substance and the like that are well known in the art. However, it is more preferable to apply an optical detection method such as surface plasmon resonance (SPR), elliptical polarization (hereinafter referred to as ellipsometry), sum frequency generation (hereinafter referred to as SFG) spectrometry or the like. Among these, SPR is particularly preferable because it is not necessary to obtain a phase difference, and a change in film thickness on the order of nm can be obtained only by obtaining a reflected light intensity. The SPR imaging method is more preferable in that a wide range of observation is possible and substance interaction observation in an array format is possible.

Phosphorylation of peptides can be performed by applying a test sample that may have protein kinases and a nucleoside triphosphate such as ATP onto the array of the present invention. Optimum phosphorylation reaction conditions vary depending on the type of protein kinase. For example, a test sample that can have protein kinase in a buffer and a nucleoside triphosphate are added, preferably at a temperature of about 10 to 40 ° C. Can be phosphorylated by reacting at a temperature of 30 to 40 ° C. for about 10 minutes to 6 hours, preferably about 30 to 1 hour. If necessary, the phosphorylation reaction solution may contain a substance that assists phosphorylation, such as cAMP, cGMP, Mg ²⁺ , and Ca ²⁺ phospholipid.

  Examples of protein kinases targeted for kinetic profiling include enzymes that phosphorylate 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.

  In the present invention, a peptide is intended to comprehensively profile the dynamics of protein kinases, so that one type of peptide is phosphorylated only by one type of protein kinase and not by other protein kinases. preferable. Peptide sequences that serve as substrates for protein kinases are known or can be appropriately selected based on known sequences. If the array of the present invention immobilizes peptides of a type corresponding to a plurality of types of protein kinases whose kinetics need to be grasped, profiling of all protein kinases can be performed with one array, which is preferable. Of course, peptides corresponding to only one type of protein kinase may be immobilized on one array, and protein kinase profiling may be performed using the required number of arrays.

  Ellipsometry can measure the film thickness and refractive index from the change in the deflection state caused by the interference between the light reflected on the surface of the thin film and the 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 with respect to p-polarized light and s-polarized light. Among them, the spectroscopic ellipsometry for measuring the ellipsometry while changing the wavelength is preferable because the change in the film thickness of the surface can be detected very sensitively.

  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 wavelength-variable 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 it is useful as a very sensitive surface analysis method.

  As described above, in one particularly preferred embodiment, it is particularly preferred that the present invention comprehensively analyzes various protein kinase activities using SPR. In SPR, an evanescent wave is generated by a polarized light beam applied to a metal and oozes on the surface, excites surface plasmons which are surface waves, consumes light energy, and reduces reflected light intensity. The resonance angle at which the reflected light intensity is significantly reduced varies depending on the thickness of the layer formed on the metal surface. Therefore, the substance or group of substances to be examined is fixed on the surface of the metal, and the interaction with the substance or group of substances in the sample is detected by changing the resonance angle or the reflected light intensity at a certain angle. Is possible. Therefore, the SPR is useful as a quantitative method that does not require labeling with a fluorescent substance, a radioactive substance, etc., and that enables real-time evaluation.

  The SPR imaging method using this SPR is a method of monitoring the state of interaction between substances by irradiating a polarized light beam over a wide range and analyzing the reflected image by making full use of image processing technology or the like. 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.

  In the SPR imaging method, in order to analyze the reflected image, a means for irradiating the chip with a polarized light beam in a wide range and sufficiently securing the illuminance of the light beam is required. An example is shown in FIG. The brighter the illuminance of the polarized light flux, the more preferable is the sensitivity of the sensor.

  The type of the light source is not particularly limited, but it is preferable to use light including near infrared light that makes the change in the SPR resonance angle particularly sensitive. Specifically, a white light source capable of irradiating 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 that is sufficiently high, has a simple light power supply and is inexpensive is particularly preferable.

  A normal white light source has a defect that light and dark unevenness occurs in the filament portion. If the light from the light source is irradiated as it is, bright and dark unevenness occurs in the image obtained by reflection, and it becomes difficult to evaluate screening and morphological changes. Therefore, as a means for uniformly irradiating the chip with light, a method of making the light into parallel light after passing through the pinhole is preferable. The means for passing the pinhole is preferable as a means for obtaining a light beam with uniform brightness, but there is a disadvantage that the illuminance is lowered when the light is passed through the pinhole as it is. Therefore, as a means for ensuring sufficient illuminance, it is preferable to use a method in which a convex lens is installed between the pinhole and the light source, and the light is collected and passed through the pinhole.

Since the white light source is radiated light, it needs to be converted into parallel light using a convex lens before condensing. Parallel light can be obtained by installing a light source near the focal length of the convex lens. By installing another convex lens and installing a pinhole in the vicinity of the focal length of the lens, it is possible to pass the condensed light through the pinhole. The light that intersects and passes through the pinhole is converted into parallel light by the CCTV lens for the camera. The cross-sectional area of the parallel light beam obtained at this time is preferably adjusted to 10 to 1000 mm ² . This method enables a wide range of screening and morphological observation.

  When the interaction is monitored, the polarized light beam is applied to the opposite surface of the metal thin film on which the substance or the substance aggregate is fixed. The polarized light beam is applied to the opposite surface of the metal thin film on which the substance or substance aggregate is fixed, and the reflected light beam is obtained. The reflected light beam from the metal thin film passes through a near-infrared wavelength optical interference filter and transmits only light in the vicinity of a certain wavelength before being photographed by a CCD camera.

  The center wavelength of the optical interference filter is preferably 600 to 1000 nm with high SPR sensitivity. The wavelength width of the wavelength at which the transmittance of the optical interference filter is half that at the maximum is called the half-value width. An image photographed by the CCD camera through the optical interference filter is taken into a computer, and a change in the brightness of a certain part can be evaluated in real time, and the whole image can be evaluated by image processing. Thus, 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.

  The chip for SPR used in the present invention preferably comprises a metal substrate in which a metal thin film is formed on a transparent substrate, and directly or indirectly, chemically or physically on the metal thin film, a substance or a substance. A slide on which the assembly 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 or plastics such as polyethylene terephthalate (PET), polycarbonate, and acrylic. Of these, glass is particularly preferable.

The thickness of the substrate is preferably about 0.1 to 20 mm, and more preferably about 1 to 2 mm. In order to achieve the object of evaluating the reflection image from the metal thin film, the SPR resonance angle is as small as possible, and there is no fear that the image to be photographed will be fuzzy, and the analysis is easy. Therefore, the refractive index n _D of the transparent substrate or the transparent substrate and the prism in contact with the transparent substrate is preferably 1.5 or more.

  Examples of the metal constituting the metal thin film include gold, silver, copper, aluminum, platinum, and the like. These may be used alone or in combination, but it is particularly preferable to use gold. The method for forming the metal thin film is not particularly limited, and examples of the known method include a vapor deposition method, a sputtering method, and an ion coating method. Of these, the method by vapor deposition is preferred. The thickness of the metal thin film is preferably about 10 to 3000 mm, more preferably about 100 to 600 mm.

  One particularly preferred embodiment of the present invention is that at least two or more peptides serving as protein kinase substrates are immobilized on a metal-deposited substrate, and the types of amino acids phosphorylated in the written peptide are different. Using an array in which two or more types exist, and a solution containing a kinase such as a cell disruption solution is allowed to act on the array, and further a chelate compound is allowed to act to determine the state of their interaction, particularly by SPR or SPR imaging. It is characterized by detecting. In the present invention, the peptide serving as a substrate for protein kinase refers to a peptide having a property of undergoing phosphorylation by the protein kinase.

  The length of the peptide is not particularly limited, but generally a peptide having 100 amino acid residues or less is used. Preferably, those consisting of 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 may be a peptide produced by a genetic engineering technique. In order to facilitate the desorption to the substrate, biotin, a cysteine residue having a thiol group added to one end of the peptide, 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.

  The method for immobilizing the peptide on the metal thin film is not particularly limited, but it is more preferable to immobilize the substrate peptide by introducing a functional group that can be easily immobilized on the surface of the metal thin film in advance. 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 onto the surface of the metal thin film, it is preferable to use a generally used derivative of alkanethiol.

  At that time, J.H. M.M. See by Brockman et al. Am. Chem. Soc. Based on the method reported in Volume 121, pages 8044-8051 (1999), a method of immobilizing through an alkanethiol layer and modifying the background with PEG (polyethylene glycol) may be used. Good. In addition, in order to further suppress non-specific effects, it is also possible to immobilize a peptide after binding a derivative in which a functional group as described above is introduced to the end of PEG to alkanethiol, which also exerts a spacer effect. It is useful in.

  Specifically, for example, carboxymethyl dextran or a water-soluble polymer such as PEG having a terminal modified with a carboxyl group is bonded to a metal thin film to introduce a carboxyl group on the surface, and EDC (1-ethyl-3, Examples of NHS (N-hydroxysuccinimide) esters using water-soluble carbodiimides such as 4-dimethylaminopropylcarbodiimide) include reacting amino groups of peptides or proteins with activated carboxyl groups. Alternatively, the surface may be modified with maleimide and then 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 the non-specific influence, the latter immobilization method via a thiol group is more preferable, but it is not particularly limited.

  A method of immobilizing a peptide to which a tag such as His-tag or GST described above is added is also very simple and useful. In this case, it is preferable to introduce NTA (nitrilotriacetic acid) and glutathione on the metal thin film after introducing an amino group or a carboxyl group to the metal surface via alkanethiol as described above. In the case of His-tag, the substrate is immobilized after the NTA-introduced array is treated with nickel chloride.

  In the present invention, a chelate compound is used in order to specifically monitor the phosphorylation of the substrate on the array with high sensitivity. The chelate compound generally refers to a complex formed by coordination of a polydentate ligand or a chelating reagent to a metal ion, and particularly a compound having a property of selectively and reversibly binding to phosphoric acid, More preferably, a polyamine zinc complex is used. More preferably, a dinuclear zinc complex having a polyamine compound as a ligand is used. Furthermore, it is more preferable to use a hexaamine dinuclear zinc (II) complex having a dinuclear zinc (II) complex in the basic structure.

A typical example of such a compound is 1,3-bis [bis (2-pyridylmethyl) amino] -2-hydroxypropanolate (IUPAC name: 1,3-bis [ bis (2-pyrylmethyl) amino] -2-propanolatodiincinc (II) complex) as a ligand is a dinuclear zinc complex (provided that the hydroxyl group of the propanol skeleton is an alcoholate with two zinc divalent ions) However, the present invention is not particularly limited to this compound.

The complex used in the present invention can be synthesized using a general chemical synthesis technique, but can also be synthesized using a commercially available compound as a raw material. For example, the compound (Zn ₂ L) represented by the above formula (I) is obtained by the following method using commercially available 1,3-bis [bis (2-pyridylmethyl) amino] -2-hydroxypropane and zinc acetate as raw materials. Can be synthesized. To a ethanol solution (100 ml) of 4.4 mmol of 1,3-bis [bis (2-pyridylmethyl) amino] -2-hydroxypropane was added 10M aqueous sodium hydroxide solution (0.44 ml), and then zinc acetate dihydrate. (9.7 mmol) is added. A brown oil can be obtained by distilling off the solvent under reduced pressure. To this residue was added 10 ml of water and dissolved, then 1M aqueous sodium perchlorate solution (3 equivalents) was added dropwise while heating to 70 ° C., and the precipitated colorless crystals were collected by filtration and dried by heating. (I) two perchlorate acetate ion adduct _{(Zn 2 L-CH 3 COO} - · 2ClO 4 - · H 2 O) represented by the structural formula can be obtained in high yield. This crystal contains one molecule of crystal water.

  In the present invention, the polyamine zinc complex as described above is modified with biotin. It is preferably modified with biotin via a linear linker structure. Specifically, the structure shown in the formula (II) is exemplified, but not particularly limited.

  The solution concentration of the biotin-modified polyamine zinc complex acting in the present invention is not particularly limited, but is usually in the range of 1 μM to 10 M, preferably 10 μM to 1 M, more preferably 10 μM to 10 mM. The mode of action on the array is not particularly limited, but the amount of liquid necessary for spreading the biotin-modified polyamine zinc complex solution over the entire array surface may be dropped, or the array may be immersed in the solution. Or you may make it act by making a solution contact on an array surface, sending a solution using a pump. The working temperature may be room temperature or may be incubated at about 20 to 40 ° C. The action time is preferably about 10 minutes to 2 hours, more preferably about 30 minutes to 1 hour.

  In the present invention, it may be possible to detect phosphorylation only by allowing a biotin-modified polyamine zinc complex to act, but in particular, by using a biotin-modified one, avidin or streptavidin is allowed to act further. As a result, it is possible to expect an effect that the detection sensitivity is further increased. It is more preferable to make streptavidin act. The concentration of avidin or streptavidin to be actuated is not particularly limited, but is usually in the range of 1 μM to 10 M, preferably 10 μM to 1 M, more preferably 10 μM to 10 mM. The mode of action on the array is not particularly limited, and is the same as in the case of the biotin-modified polyamine zinc complex.

  In a further preferred embodiment, after avidin or streptavidin is allowed to act, an antibody that recognizes avidin or streptavidin is allowed to act further, so that the detection sensitivity can be further enhanced. The concentration at which the antibody is allowed to act is not particularly limited, but is preferably about 0.01 to 10 μg / ml, more preferably about 0.1 to μg / ml. As the antibody, either a monoclonal antibody or a polyclonal antibody can be applied, but the monoclonal antibody is preferred in terms of specificity. The mode of action on the array is also not particularly limited, and is the same as in the case of biotin-modified polyamine zinc complex, avidin or streptavidin.

  Further, a biotin-modified polyamine zinc complex, avidin or streptavidin may be allowed to act sequentially, but a biotin-modified polyamine zinc complex and a complex of avidin or streptavidin previously formed may be allowed to act directly. In this case, as described above, an antibody that recognizes avidin or streptavidin may be allowed to further act. In forming the complex, it is preferable that the biotin-modified polyamine zinc complex and avidin or streptavidin are reacted in a molar ratio of 1: 1 to 4: 1. The reaction product is preferably purified to remove unreacted product, but the reaction product can be applied as it is.

  The application of such a chelating compound is advantageous in that it can be synthesized at a very low cost by the method described above. In addition, it is stable and easy to use in that it can be stored at room temperature, which is advantageous in terms of distribution. Compared to the detection method using antibodies in particular, it works regardless of the type of amino acid residue to be phosphorylated, and the reaction does not depend on the amino acid sequence in the vicinity of the phosphorylated amino acid. And has a great advantage.

  Examples of the protein kinase include various tyrosine kinases and serine / threonine kinases. The types of these protein kinases are not particularly limited, and can basically be applied to all types of protein kinases.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated more concretely, this invention is not specifically limited to an Example.

[Example 1]
(Peptide immobilization)
4 armPEG (SUNBRIGHT PTE-100SH manufactured by NOF Corporation) whose terminal functional group is a thiol group was dissolved in a mixed solution of 7 ml of ethanol: water = 6: 1 at a concentration of 1 mM. The molecular weight of 4armPEG is 10,000, which is a molecule in which four PEG chains having almost the same length from the center are present, and is very hydrophilic. In addition, all four ends of PEG are thiol groups, and particularly exhibit metal binding properties to gold. A gold-deposited slide in which 3 nm of chromium was vapor-deposited on an SF15 glass slide of 18 mm square and 2 mm thickness and gold was vapor-deposited at 45 nm was immersed in the 4 armPEG thiol solution for 3 hours to bond 4 armPEG thiol to the entire gold substrate.

  A photomask was placed on the slide and irradiated with a 500 W ultra-high pressure mercury lamp (manufactured by USHIO) for 2 hours to remove 4 armPEG thiol in the UV irradiation part. The photomask has 96 square holes of 500 μm square (consisting of 8 × 12 patterns), and the pitch between the hole centers is designed to be 1 mm. The portion of the photomask with a hole is transparent to UV light and is irradiated onto the slide for patterning. 4 armPEG remains in the part that was not irradiated and functions as a reference part as a background part of the chip.

  It was immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 1 hour to form a self-organized surface of 8-AOT on the UV irradiation part. A heterobifunctional polyethylene glycol (NHS-PEG-MAL, manufactured by Nektar) having a succinimide (NHS) group and a maleimide (MAL) group at the end of a molecular weight of 3400 is phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7. 2) was dissolved at 10 mg / ml and reacted with 8-AOT on the gold surface for 2 hours. Since the amino group of 8-AOT and the NHS group of NHS-PEG-MAL reacted and the MAL group remained unreacted, a maleimide group could be introduced to the surface via PEG.

Five types of protein kinase substrates (phosphorylated substrate and non-phosphorylated substrate) were immobilized on the surface obtained as described above. The amino acid sequence of each substrate peptide and the arrangement regarding its immobilization are shown in the lower part of FIG. A blank indicates a blank spot where the peptide is not immobilized. Each substrate peptide was dissolved in a phosphate buffer solution (20 mM phosphate, 150 mM NaCl; pH 7.2) at 1 mg / ml, and spotted 10 nl at a time using a MultiSPRinter ^™ spotter (manufactured by Toyobo). Then, the immobilization reaction was carried out by allowing to stand at room temperature for 16 hours in a wet environment. The maleimide group formed on the surface of the chip reacts with the thiol group possessed by the cysteine residue at the terminal of the substrate peptide, so that the substrate peptide can be covalently immobilized on the surface.

(Blocking of unreacted maleimide groups)
After the surface on which the substrate peptide is immobilized is washed with a phosphate buffer, in order to block unreacted maleimide groups, PEG thiol (SUNBRIGHT MESH-50H manufactured by NOF Corporation) is phosphate buffer so that the concentration becomes 1 mM. Dissolved in (20 mM phosphoric acid, 150 mM NaCl; pH 7.2), 300 μl was poured onto the chip and allowed to react at room temperature for 30 minutes. The molecular weight of PEG thiol used here is 5,000.

(Detection of phosphate groups on the array)
The array that had been blocked as described above was washed with PBS and water to allow the biotin-modified polyamine zinc complex to act. As the biotin-modified polyamine zinc complex, Phos-tag ^™ BTL-104 (purchased from Nard Laboratory Co., Ltd.) represented by the following formula (II) was used. Phos-tag ^™ BTL-104 had a concentration of 25 μg / ml, and the lysis solution contained 10 mM HEPES-NaOH buffer containing 0.005% Tween 20, 10% (v / v) ethanol, 0.2 M sodium nitrate, 1 mM zinc nitrate. (PH 7.4) was used. The action was carried out for 1 hour at room temperature.

The above-treated array was washed with PBS and water and set in an SPR apparatus (MultiSPRinter ^™ manufactured by Toyobo Co., Ltd.) for analysis. As the running buffer, the same 0.005% Tween 20, 10% (v / v) ethanol, 10 mM HEPES-NaOH buffer (pH 7.4) containing 0.2 M sodium nitrate and 1 mM zinc nitrate was used. Streptavidin (manufactured by Molecular Probes) is dissolved in the same buffer to prepare solutions of 1, 5, 10, 50 μg / ml concentration, and arrayed while sequentially feeding using a planger pump (Model 021 manufactured by Flume). Acted on the surface. The temperature was set at 30 ° C. The obtained sensorgram is shown in FIG. Although there is a difference in strength depending on the type of phosphorylated substrate, a significant signal increase can be confirmed in any phosphorylated substrate as compared to the non-phosphorylated substrate.

The results of SPR imaging are shown in the upper part of FIG. In this SPR analysis, an image is captured every 5 seconds by a CCD camera. From an image captured at the time after the streptavidin (hereinafter also referred to as SA) reaction, an image at the time before the reaction is obtained. Is a result of performing a subtraction process using image calculation processing software Scion Image (manufactured by Scion Corp.). Spots are observed only at the site where the phosphorylated substrate is immobilized, and it is confirmed that Phos-tag ^™ BTL-104 is specifically bound.

[Example 2]
The same array as in Example 1 was used until blocking of the unreacted maleimide group, and the array was washed with PBS and water, and set in an SPR device (MultiSPRinter ^™ manufactured by Toyobo Co., Ltd.) for analysis. . The same running buffer as in Example 1 was used. Phos-tag ^™ BTL-104 is dissolved in running buffer to prepare solutions of 1,5,10 μg / ml concentration, and it is applied to the surface of the array while being sequentially fed using a planger pump (Model 021 manufactured by Flume). I let you. The temperature was set at 30 ° C. Thereafter, washing was performed while the running buffer was fed, and then the streptavidin solution was fed in the order of 1, 5, 10 μg / ml concentration to act. The obtained sensorgram is shown in the upper part of FIG. Even in this case, although the intensity varies depending on the type of phosphorylated substrate, a significant signal increase can be confirmed in any phosphorylated substrate compared to the non-phosphorylated substrate. The results of SPR imaging performed in the same manner as in Example 1 are shown in the lower part of FIG. The same tendency is confirmed for this.

[Example 3]
(Peptide immobilization)
Binding of 4armPEG thiol to the gold substrate was performed in the same manner as in Example 1. In the subsequent UV irradiation, patterning was performed in the same manner as in Example 1 except that a photomask having 16 square holes of 500 μm square (consisting of 4 × 4 patterns) was used. went. Subsequent introduction of amino groups onto the array surface and formation of maleimide group surfaces using a crosslinking agent were carried out in the same manner as in Example 1. The spotting of the substrate peptide was performed by 0.1 μl by manual operation. As a substrate, a PKA (protein kinase A) substrate (same as PKA (Ser) in FIG. 2), a positive control (pPKA) in which the serine residue is phosphorylated in advance, and the serine residue is substituted with an alanine residue Using a negative control (nPKA), the cells were immobilized in the arrangement as shown in the lower part of FIG. The reaction after spotting was performed in the same manner as in Example 1.

(Blocking of unreacted maleimide groups)
The same procedure as in Example 1 was performed.

(Detection of phosphate groups on the array)
The array that had been blocked as described above was washed with PBS and water, and phosphorylated with PKA. 400 μl of PKA solution was dropped on the array and reacted at 30 ° C. for 30 minutes. The composition of the PKA solution was 1 μl of PKA catalyst subunit (manufactured by Promega), 375 μl of 50 mM Tris-HCl buffer (pH 7.4), 20 μl of 1M magnesium chloride solution, and 4 μl of 10 mM ATP.

The array subjected to the PKA reaction was washed with PBS and water, and Phos-tag ^™ BTL-104 was allowed to act under the same conditions as in Example 1. The array was washed with PBS and water, and set in an SPR device (MultiSPRinter ^™ manufactured by Toyobo Co., Ltd.) for analysis. The same running buffer as in Example 1 was used. In the same manner as in Example 1, the streptavidin solution was allowed to act while being fed in the order of 1, 5, 10 μg / ml concentration. The resulting sensorgram and the results of SPR imaging are shown in FIG. In the positive control, the strongest binding signal was confirmed, and in the PKA substrate, the binding signal was confirmed to some extent. There was almost no signal change in the negative control and blank. This result indicates that phosphorylation of the PKA substrate on the array can be detected.

[Example 4]
The same PKA substrate as in Example 3 and its positive control and negative control and an array in which cSrc substrate and its positive control were immobilized at 96 points were prepared in the same manner as in Example 1. The arrangement of the substrate is as shown in the lower part of FIG. Blocking, PKA reaction, and Phos-tag ^™ BTL-104 were performed in the same manner as in Example 3. The array was washed with PBS and water, and set in an SPR device (MultiSPRinter ^™ manufactured by Toyobo Co., Ltd.) for analysis. Went. The same running buffer as in Example 1 was used. A streptavidin solution (10 μg / ml) was allowed to act by feeding. After confirming that the signal rise becomes a plateau, the plate was washed with a running buffer solution, and a streptavidin antibody (manufactured by Vector) was adjusted to a concentration of 2.5 μg / ml and acted on the solution. The obtained sensorgram and the result of SPR imaging are shown in FIG. In the positive control of PKA and cSrc substrate, a strong signal increase was confirmed in both cases, and a certain level of signal increase was also confirmed in the PKA substrate. There is almost no signal change in the negative control and blank. The increase in signal is more prominent than that caused by the action of a streptavidin antibody, and the specificity is also excellent, indicating that it has an excellent sensitizing effect.

[Example 5]
An array in which phosphorylated and non-phosphorylated substrates were immobilized on the PKA substrate (threonine type), PKC substrate (serine type), and cSrc substrate (Yao; tyrosine type) shown in FIG. 2 was prepared in the same manner as in Example 1. . Subsequent blocking was performed in the same manner as in Example 1 and set in an SPR device (MultiSPRinter ^™ manufactured by Toyobo). On the other hand, Phos-tag ^™ BTL-104 solution (50 μg / ml) and streptavidin solution (75 μg / ml) were mixed in equal amounts and reacted at room temperature for 30 minutes to form a complex. This complex solution was diluted 10-fold and 5-fold with the same running buffer as in Example 1 and allowed to act while being fed. Further, after confirming that the signal rise becomes a plateau, the solution was washed with a running buffer solution, and the streptavidin antibody (manufactured by Vector) was adjusted to a concentration of 2.5 μg / ml and allowed to act by feeding the solution. The obtained sensorgram is shown in FIG. In this case as well, a specific signal increase can be observed only in the phosphorylated substrate. The results of SPR imaging and the arrangement of the substrates are shown in FIG.

  According to the method of the present invention, no special technique is required, and in particular when SPR is used, there is no need to use labels for fluorescent substances, radioactive substances, etc. It became possible to analyze the dynamics. In particular, this is a useful technique for simultaneously measuring assays relating to substrates having different types of phosphorylated amino acids. By using a chelate compound, the present invention has a great advantage over conventional methods in that it is inexpensive and easy to handle and is not affected by the type of phosphorylated amino acid or the amino acid sequence in the vicinity thereof. . By using the present invention, it is possible to comprehensively analyze various types of protein kinase signals, and effectively profile protein kinase dynamics in cells accompanying the introduction of genes with unknown functions or drug administration. be able to. As a result, development to genome drug discovery such as functional analysis from new genes and approaches to new drug discovery is expected, which is also meaningful in industry.

6 is a diagram of SPR analysis in Example 1. FIG. In Example 1, it is a figure which shows the result of having performed the pattern which fixed the substrate peptide on the array, and SPR imaging. It is a figure which shows the result of having performed SPR analysis and SPR imaging in Example 2. FIG. In Example 3, it is a figure which shows the result of having performed the pattern which fixed the substrate peptide on the array, and SPR analysis and SPR imaging. It is a figure of SPR analysis in Example 4. In Example 4, it is a figure which shows the result of having performed the pattern which fixed the substrate peptide on the array, and SPR imaging. It is a figure which shows the result of having performed the SPR analysis in Example 5. FIG.

Claims (10)

  A method for detecting phosphorylation of a peptide on an array in which two or more kinds of peptides are immobilized on a metal thin film on a substrate, wherein the two or more kinds of amino acids differ as phosphorylation sites in each of the immobilized peptides A method for analyzing protein kinase activity, wherein a peptide containing a residue is immobilized, and a polyamine zinc complex modified with biotin is allowed to act upon detection of phosphorylation.   2. The protein according to claim 1, wherein an array on which two or more kinds of peptides containing amino acid residues of a combination of two or more of serine, threonine, and tyrosine are immobilized is used as a site to be phosphorylated. Analysis method of kinase activity.   3. The method for analyzing protein kinase activity according to claim 1 or 2, wherein after the polyamine zinc complex modified with biotin is allowed to act, avidin or streptavidin is further allowed to act.   The method for analyzing protein kinase activity according to any one of claims 1 to 3, wherein an antibody that recognizes avidin or streptavidin is further allowed to act after avidin or streptavidin is allowed to act.   A method for detecting phosphorylation of a peptide on 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 of a substrate, which is modified with biotin A method for analyzing protein kinase activity, comprising forming a complex of a polyamine zinc complex with avidin or streptavidin and allowing the complex to act.   6. The method for analyzing protein kinase activity according to claim 5, wherein after the complex is allowed to act, an antibody that recognizes avidin or streptavidin is further allowed to act. The method for analyzing protein kinase activity according to any one of claims 1 to 6, wherein a binuclear zinc complex having a polyamine compound as a ligand is used as the polyamine zinc complex. The method for analyzing protein kinase activity according to any one of claims 1 to 7, wherein the compound represented by formula (I) is used as the polyamine zinc complex.
The method for analyzing protein kinase activity according to any one of claims 1 to 8, wherein the phosphorylated peptide is detected using surface plasmon resonance (SPR). The method for analyzing protein kinase activity according to claim 9, wherein the phosphorylated peptide is detected by a surface plasmon resonance imaging method.