JP2006223187A - Profiling method of protein kinase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain technique for obtaining profiling data, in which influence of array preparation conditions and variable factor of lot of a substrate is almost avoided, extremely excellent in reproducibility and having high accuracy and reliability. <P>SOLUTION: The profiling method of protein kinase activity comprises analyzing protein kinase by using an array on which a plurality of peptides are immobilized and using a chelate compound by a surface plasmon resonance (SPR) analysis. In the profiling method, one or more kinds of peptides previously phosphorylated are immobilized on the array and the SPR signal intensities in various kinds of peptides immobilized after phosphorylation reaction on the array and the ratios of SPR signal intensities in the previously phosphorylated peptides are calculated and these values are compared. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for comprehensively analyzing protein kinase activity by an analysis technique using surface plasmon resonance (SPR). More specifically, the present invention relates to a method for realizing profiling with respect to protein kinase activity quantitatively to some extent by calculating a signal ratio using a substrate internal standard previously phosphorylated as an internal standard.

  In recent years, biochips used for biomolecule interaction analysis, profiling of expressed molecules, or diagnosis have attracted attention. The operation is facilitated by immobilizing the biomolecule on the substrate, and in some cases, the interaction of a very large number of substances can be analyzed. In particular, peptide arrays in which peptides with relatively small molecular weights are immobilized on a substrate have relatively few problems of denaturation such as proteins, and have strong combinatorial chemistry aspects. It has come to be widely used for searching and the like.

  Such analysis technology using a biochip is widely used as an analysis means / tool in proteomics. For example, analysis of networks related to post-translational modification of proteins, particularly protein phosphorylation, is important in understanding the life phenomena that are carried out in cells. For this purpose, there is a demand for means for comprehensively analyzing in detail including activity regulation and function regulation by phosphorylation modification. 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 signal transduction pathways do not function independently, but function as a system by cross-talking with each other. 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 sufficient level of technology capable of simultaneously profiling the dynamics of various protein kinases in a simple and efficient manner has not yet been established. Even more so far, the technology that allows the profiling results in phosphorylation on the array to be considered from a quantitative point of view is hardly realized.

As a related technique that has already been reported, it has been reported that the activity of cSrc kinase, which is a kind of tyrosine kinase, was evaluated using, for example, 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, refer to Non-Patent Documents 2 and 3), or examples for a detection system of a kinase reaction on an array using a radioactive substance ([γ ³² P] ATP) (for example, 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.

  Further, in the profiling related to phosphorylation as described above, it may be necessary to evaluate not only from a qualitative level but also from a quantitative viewpoint depending on the purpose. However, it is very difficult to simultaneously quantify how much phosphorylation each individual substrate has when arrayed, and it has been hardly realized in the techniques reported so far. Absent.

Benjamin T. Houseman et al., Nature Biotechnology Volume 20, 270-274 (issued in 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)

  An object of the present invention is to provide a useful technique for quantifying the reaction efficiency by performing simple arithmetic processing from the signal intensity due to phosphorylation obtained on the array and clarifying the analysis data of the profiling result. It is to provide.

As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means, and have reached the present invention.
1. This is a method for analyzing protein kinase activity by surface plasmon resonance (SPR) analysis using an array in which multiple types of peptides are immobilized. In detecting phosphorylation, a chelate compound modified with a ligand is used. And one or more kinds of peptides phosphorylated in advance on the array, and SPR signal intensity in the various peptides immobilized after the phosphorylation reaction on the array and the phosphorylation in advance A method for profiling protein kinase activity, comprising calculating and comparing a value of a ratio of SPR signal intensities in the obtained peptide.
2. 1. The method for profiling protein kinase activity according to 1, wherein an array in which the same peptide phosphorylated in advance is immobilized at two or more sites is used.
3. A method for profiling protein kinase activity according to 1 or 2, wherein a chelate compound modified with a ligand is allowed to act and then a receptor is allowed to act further.
4). The method for profiling protein kinase activity according to any one of 1 to 3, wherein an antibody that recognizes the receptor is further reacted after the receptor is allowed to act.
5. The method for profiling protein kinase activity according to any one of 1 to 4, wherein the chelate compound modified with a receptor has a molecular weight of 500 to 1,000.
6). The method for profiling protein kinase activity according to any one of 1 to 5, wherein the ligand is biotin and the receptor specific for the ligand is avidin or streptavidin.
7). The method for profiling protein kinase activity according to any one of 1 to 6, wherein the chelate compound is a polyamine zinc complex.
8). The method for profiling protein kinase activity according to any one of 1 to 7, wherein a binuclear zinc complex having a polyamine compound as a ligand is used as a chelate compound.
9. The method for profiling protein kinase activity according to any one of 1 to 8, wherein the compound described in formula (I) is used as a chelate compound.
10. The method for profiling protein kinase activity according to any one of 1 to 9, wherein the peptide is immobilized via a thiol group.
11. The method for profiling protein kinase activity according to any one of 1 to 10, wherein the number of peptides to be immobilized is 24 or more.
12 The method for profiling protein kinase activity according to any one of 1 to 11, wherein the number of peptides to be immobilized is 48 or more.
13. The method for profiling protein kinase activity according to any one of 1 to 12, wherein the number of peptides to be immobilized is 96 or more.

In the protein kinase activity profiling method of the present invention, the ratio of the SPR signal intensity in various immobilized peptides after the phosphorylation reaction on the array and the SPR signal intensity in the previously phosphorylated peptide are calculated and compared. By doing so, the problem that the absolute value of the SPR signal intensity varies depending on the production lot of the array and the number of measurements can be solved, and the results of clear and effective profiling analysis can be solved. Obtainable.

  The method for immobilizing a peptide on a substrate in the present invention is not particularly limited, and examples thereof include a method for immobilizing a peptide via a thiol group. The peptide used here is exemplified by a generally used meaning, but may be a high molecular weight protein. This is one in which two or more amino acid residues are linked by peptide bonds and has the property of functioning as a protein kinase substrate. The number of amino acid residues in the substrate sequence site is not particularly limited, but is usually about 5 to 60 residues, and more preferably about 10 to 25 residues. Depending on the purpose, amino acids in which one to several residues among the amino acid residues are chemically modified may be included.

  Here, the site immobilized on the substrate specifically refers to a state in which at least one cysteine residue is present in the amino acid sequence of the peptide to be immobilized. The cysteine residue is an amino acid necessary for the peptide to be immobilized to have its original function, even if it exists as an essential residue as an amino acid sequence necessary for the peptide to function. It may be a case where it is further added to the sequence. The position of the cysteine residue in the peptide to be immobilized is not particularly limited, but it is preferably added to at least one end, more preferably only to one end. As another embodiment, a compound in which a compound having a thiol group is chemically bonded to one or more amino acid residues with respect to the peptide to be immobilized. In this case, the bonding position of the compound is not particularly limited, but is preferably bonded to any terminal amino acid residue.

  It is preferable that a hydrophilic compound is inserted in advance as a spacer between the site immobilized on the substrate and the substrate sequence site. Although the molecular weight of a hydrophilic compound is not specifically limited, About 100-1000 are preferable and 400-1000 are more preferable. In addition to the hydrophilic compound, a spacer sequence having 2 to 10 amino acid residues, more preferably 2 to 6 amino acid residues may be further added. The type of amino acid residue constituting the spacer sequence is not particularly limited, but it is preferable to include an amino acid that is unlikely to form a higher order structure. Specifically, at least one preferably contains a glycine residue. More preferably, one residue of glycine (G), two residues of glycine (GG), glycine and alanine (GA or AG), or glycine and serine (GS or SG) residues are repeated one or more times. A sequence comprising two or more times is exemplified.

  When immobilizing a peptide via a thiol group as described above, an amino group is introduced into the substrate surface in advance, and a heterobifunctional crosslinking agent having a succinimide (NHS) group and a maleimide (MAL) group is used. A method of reacting by forming a maleimide surface is recommended. The means for introducing an amino group on the chip is not particularly limited. Examples thereof include a self-assembled surface method for aligning molecules on the substrate surface, a method of introducing using a reaction reagent, and a means for coating a substance having a functional group on a chip. Also included are means for introducing an amino group using a cross-linking agent starting from the functional group introduced on the surface. Such heterobifunctional crosslinking agents include various commercially available ones, and are not particularly limited. For example, succinimidyl 4- [N-maleimidemethyl] cyclohexane-1-carboxylate (hereinafter referred to as SMCC). Alternatively, sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (hereinafter referred to as SSMCC) can be used. In addition, even if these compounds are not completely the same in structure, analogized compounds can be applied as long as their functions are not impaired. Either SMCC or SSMCC can be applied, but from the viewpoint of solubility in water, it is more preferable to use SSMCC when the reaction is carried out in an aqueous system such as a buffer solution. In addition, those having a succinimide (NHS) group and a maleimide (MAL) group at the end of a polymer such as PEG can also be used.

  In the label-free optical detection method, any substance adsorbed on the chip is detected as a signal. That is, it is difficult to distinguish nonspecific adsorption of a substance that is not a measurement target from specific adsorption. Therefore, since a means for suppressing non-specific adsorption more severely is required, the above-described immobilization method is very effective.

  In an ELISA method or an interaction analysis method using a label substance, physical adsorption by bovine serum albumin or casein is generally selected as a blocking method. Although the physical adsorption method is easy, it is not stable and may be detached from the chip surface over time. In the above optical detection method, it is preferable to perform blocking by covalent bond in order to detect even the elimination of the blocking agent. In particular, when blocking the unreacted maleimide group surface, a compound having a thiol group is preferably used, and a PEG derivative is particularly preferably used.

  In SPR, a metal substrate is used. In the present invention, the material of the substrate is preferably gold which is very stable in acid, alkali, organic solvent and the like. In fact, gold is a material frequently used in the above optical detection method. Further, the material supporting gold is preferably transparent, and more preferably transparent glass. This is because transparent glass is not only easily available, but also very suitable for SPR measurement.

  As a method of forming the metal substrate, a method of coating a thin metal layer is preferable. The method for coating the metal is not particularly limited, but generally, a vapor deposition method, a sputtering method, an ion coating method, or the like is selected. In order to provide an optical detection method, it is necessary to control the thickness of the thin metal layer at the nano level. The thickness of the thin metal layer is not particularly limited, but is generally selected in the range of 30 nm to 80 nm. In order to suppress peeling of the thin metal layer, a chromium layer or a titanium layer of 0.5 nm to 10 nm may be coated on the substrate in advance.

  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. Yes, 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. 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.

One particularly preferred embodiment of the present invention uses an array in which a plurality of peptides serving as protein kinase substrates are immobilized on a metal-deposited substrate, and the array contains a kinase such as a cell lysate. It is characterized by detecting the state of their interaction by applying a solution, particularly by SPR or SPR imaging. 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. The phosphorylation reaction may be performed by dropping and incubating a solution containing or presumed to contain protein kinase on the array, or by using a liquid feeding means such as a pump, the array is placed in the SPR device. You may go while setting. If necessary, the phosphorylation reaction solution may contain a substance that assists phosphorylation, such as cAMP, cGMP, Mg ²⁺ , and Ca ²⁺ phospholipid. In the detection of phosphorylation, the uptake of phosphorus may be confirmed directly, or it is detected by the action of an antibody that recognizes phosphorylated amino acids or a substance that has a specific binding property to a phosphate group. May be.

  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 chelate compound modified with a ligand is allowed to act in order to monitor the phosphorylation of the substrate on the array with high sensitivity. A chelate compound generally refers to a complex formed by coordination of a polydentate ligand or chelating reagent to a metal ion such as zinc, iron, cobalt, palladium, or zirconium, but is particularly selective for phosphoric acid. Further, a compound having a property of reversibly binding is preferable, and a polyamine zinc complex is more preferably 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.

  The ligand and receptor used in the present invention are preferably selected to have a relationship capable of specifically recognizing and binding to each other. Examples of ligands and receptors include biotin and avidin, biotin and streptavidin, steroid hormones and steroid hormone receptors, nucleic acids and transcription factors, a single-stranded nucleic acid sequence and a single strand having a sequence complementary to the single-stranded nucleic acid sequence. Examples include strand nucleic acid sequences. Among these, it is preferable to use biotin as a ligand and avidin or streptavidin as a receptor, but it is not limited thereto.

  In the present invention, the polyamine zinc complex as described above is modified with a ligand such as biotin, for example. The ligand is not limited to biotin, and may be a steroid hormone or a nucleic acid. It is preferably modified with biotin via a linear linker structure. And as the molecular weight, it is the range of 500-1000, More preferably, it is 600-900, More preferably, it is 700-900. Specifically, the structure shown in the formula (II) is exemplified, but not particularly limited. When the molecular weight of the biotin-modified polyamine zinc complex exceeds 1000, the stability is also deteriorated, which is not preferable from the viewpoint of the binding efficiency to phosphoric acid.

  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, for example, avidin or streptavidin By acting such a receptor, it can be expected that the detection sensitivity is further increased. The receptor is not limited to avidin or streptavidin, and may be a steroid hormone receptor, a transcription factor, a nucleic acid, or the like. 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 particularly advantageous in that it can be synthesized at a very low cost as compared with conventionally used antibodies. 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 with the detection method using antibodies in particular, it acts regardless of the type of amino acid residue to be phosphorylated and is independent of the reaction with the amino acid sequence in the vicinity of the phosphorylated amino acid. Have great advantages.

  On the array used in the present invention, it is preferable that substrate peptides consisting of as many amino acid sequences as possible are immobilized. The number is not particularly limited, but is preferably 24 or more, more preferably 48 or more, and still more preferably 96 or more. Of course, it may be several hundred to thousands or more.

  In the profiling method of the present invention, one or more kinds of peptides phosphorylated in advance on the array are immobilized, and the SPR signal intensity in the various peptides immobilized after the phosphorylation reaction on the array is determined in advance. The SPR signal intensity ratio value in the phosphorylated peptide is calculated and compared. The peptide phosphorylated in advance is not particularly limited, and may be one kind or plural kinds. Moreover, although it may be only one place or may be a plurality of places, it is preferably a form in which a plurality of places are fixed in a random arrangement as much as possible. In that case, it is preferable to use a value obtained by averaging the signal intensities of all phosphorylated substrates. As a more preferred embodiment, it is conceivable to use a peptide in which the phosphorylation site is phosphorylated in advance with an amino acid sequence that is known to be phosphorylated to some extent reliably by the protein kinase to be acted on. The phosphorylation site is a serine, threonine or tyrosine residue.

  After performing the SPR analysis after the phosphorylation reaction, the ratio of the SPR signal intensity in the immobilized various peptides and the SPR signal intensity in the above-mentioned previously phosphorylated peptide is calculated and compared. Arithmetic processing can be easily performed by using commercially available software. Thus, by using the ratio with the SPR signal intensity in the peptide phosphorylated in advance, it is possible to solve the big problem in quantification that the signal intensity varies due to the difference in the production lot of the array or the difference in the measurement day. it can. By performing profiling by performing such calculation processing, profiling data with extremely high reproducibility and high reliability can be obtained with little influence from variable factors such as array fabrication conditions and substrate lots. Is something that can be done. As a result, it is possible to discuss the phosphorylation efficiency from a quantitative viewpoint. In addition, since the comparison of phosphorylation rates between substrates is easy and clear, it is a useful technique even when, for example, screening and selection of a substrate superior to a known substrate is performed. Based on this theory, software for organizing and analyzing profiling data related to phosphorylation can also be developed.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not particularly limited to the examples.

[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 approximately 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. SSMCC (Pierce) was dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 0.4 mg / ml and reacted with 8-AOT on the gold surface at room temperature for 15 minutes. The amino group of 8-AOT and the NHS group of SSMCC reacted, and the MAL group remained unreacted and could be introduced onto the surface.

  Immobilization was performed by spotting 171 types of peptides shown in the sequence listing on the surface obtained as described above. Among the 171 substrates, there are four control peptides, known PKA substrate (SEQ ID NO: 69), PKC substrate (SEQ ID NO: 70) and two cSrc kinase substrates (SEQ ID NO: 71, 72). As shown in FIG. 1, immobilization was carried out by dividing into two arrays. No. 1, no. In both arrays of 2, the peptide in which serine was previously phosphorylated in the PKA substrate of SEQ ID NO: 69 was immobilized at 8 positions as shown in the figure as an internal standard. Also, for any peptide, a cysteine residue is added to the terminal, and a spacer sequence of glycine 2 residues exists between the cysteine residue and the substrate sequence.

  All the above peptides were dissolved in a phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 1 mg / ml, and a peptide solution was 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 washing the substrate peptide-immobilized surface with a phosphate buffer, in order to block unreacted maleimide groups, PEG thiol (the functional group at one end is a thiol group and the other functional group is a methoxy group) NOF SUNBRIGHT MESH-50H) is dissolved in a phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) to a concentration of 1 mM, 250 μl is poured onto the chip, and allowed to react at room temperature for 30 minutes. It was. The molecular weight of PEG thiol used here is 5,000.

(Detection of phosphorylation by PKA by SPR analysis)
Using the array that had been blocked as described above, phosphorylation was performed on-chip with PKA. 400 μl of PKA solution was dropped on the array and reacted at 30 ° C. for 4 hours. The composition of the PKA solution was 1 μl of cAMP-dependent protein kinase catalytic unit (Promega; 80 U / μl), 275 μl of 50 mM Tris-hydrochloric acid buffer (pH 7.4), 20 μl of 1M magnesium chloride solution, 4 μl of 10 mM ATP. Thereafter, the array was washed three times with PBS and water, the array surface was dried, and then a biotin-modified polyamine zinc complex was allowed 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 has a concentration of 25 μg / ml, and the lysis solution contains 0.005% Tween 20, 10% (v / v) ethanol, 0.2 mM sodium nitrate, 10 mM HEPES-NaOH buffer containing 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. The running buffer was flowed into the flow cell at a rate of 100 μl / min using a planner pump (Model-021 manufactured by Flume). After confirming that the signal from the SPR was stabilized, streptavidin (manufactured by Molecular Probes) was dissolved in the same buffer to prepare a solution having a concentration of 10 μg / ml and allowed to act on the array surface while being fed. The temperature was set at 30 ° C. When the signal rise reached a plateau state, the running buffer was sent again for washing, and a 2 μg / ml streptavidin antibody solution was similarly prepared for action. When the signal rise reached a plateau state, the running buffer was fed again for washing. The change of the SPR signal at that time was observed. In addition to the spot site of each substrate, the signal change was also observed in the background.

(Observation results and discussion)
The results of SPR imaging are shown in FIG. During the SPR analysis, an image is captured by a CCD camera every 5 seconds. From an image captured at the time after the antibody reaction, an image at the time before the reaction is used as an image calculation processing software as a scion image (manufactured by Scion Corp.). ) Is the result of performing the subtraction process. A strong increase in antibody binding signal has been confirmed against 8 phosphorylated PKA substrates which are internal standards. In addition, regarding the PKA substrate of SEQ ID NO: 69 as a control, phosphorylation signals could be confirmed in both arrays.

  Also, the increase in SPR signal after the antibody action in the above-mentioned 8 phosphorylated PKA substrates in each array was averaged, and the ratio between the average value and the increase in SPR signal in each immobilized peptide was calculated. Calculated. The results of graphing the results are shown in FIG. It has been confirmed that some of the substrates show a fairly strong signal even when compared with the Kemptide substrate (SEQ ID NO: 69), which has been widely known as a substrate for PKA. It is believed that there is.

[Example 2]
Using the same array as in Example 1, the steps up to the blocking treatment were performed in the same manner, and phosphorylation with PKCβI was performed on-chip. The reaction was carried out at 30 ° C. for 1 hour. The reaction solution composition was PKCβI (manufactured by Sigma) with the same buffer composition as in Example 1, and further 0.1 mM calcium chloride, 8 μg / mL phosphatidylserine (manufactured by Sigma), 0.8 μg / mL. Concentration of diacylglycerol (Sigma) was added. FIG. 4 shows the results of SPR imaging using a zinc chelate compound, streptavidin, and streptavidin antibody reaction by the same operation as in Example 1 using the array after phosphorylation. Further, the results of similar profiling analysis are shown in FIG.

  4 and 5 show that some substrates show a fairly strong signal. It has been confirmed that the phosphorylation pattern is greatly different from that in the case where the phosphorylation reaction by PKA is performed. In particular, it can be seen that the difference is clearer when comparing FIG. 3 and FIG. It has also been confirmed that the reproducibility regarding this result is very good.

  By the method of the present invention, the influence of variation factors such as array fabrication conditions and substrate lots is almost avoided, and from a quantitative point of view, highly accurate and highly reliable profiling data with excellent reproducibility regarding phosphorylation patterns can be obtained. It can be obtained. By obtaining a phosphorylation pattern based on this theory, it is expected to greatly contribute to various industries including drug discovery screening.

In Example 1 and Example 2, it is a figure which shows the fixed position of the substrate peptide in the produced array for SPR. In Example 1, it is a figure which shows the result of having performed SPR imaging regarding the on-chip phosphorylation reaction by PKA. In Example 1, it is a figure which shows the result of having performed the profiling analysis process by the method of this invention regarding the on-chip phosphorylation reaction by PKA. In Example 2, it is a figure which shows the result of having performed SPR imaging regarding the on-chip phosphorylation reaction by PKC (beta) I. In Example 2, it is a figure which shows the result of having performed the profiling analysis process by the method of this invention regarding the on-chip phosphorylation reaction by PKC (beta) I.

Claims (13)

  This is a method for analyzing protein kinase activity by surface plasmon resonance (SPR) analysis using an array in which multiple types of peptides are immobilized. In detecting phosphorylation, a chelate compound modified with a ligand is used. And the SPR signal intensity in each of the various peptides immobilized after the phosphorylation reaction on the array and the phosphorylation in advance. A method for profiling protein kinase activity, comprising calculating and comparing a value of a ratio of SPR signal intensities in the obtained peptide.   2. The method for profiling protein kinase activity according to claim 1, wherein an array in which the same peptides phosphorylated in advance are immobilized at two or more sites is used.   3. The method for profiling protein kinase activity according to claim 1 or 2, further comprising the step of allowing a receptor to act after acting a chelate compound modified with a ligand.   4. The method for profiling protein kinase activity according to any one of claims 1 to 3, wherein after the receptor is acted, an antibody which recognizes the receptor is further acted.   The method for profiling protein kinase activity according to any one of claims 1 to 4, wherein the chelate compound modified by the receptor has a molecular weight of 500 to 1,000.   6. The method for profiling protein kinase activity according to any one of claims 1 to 5, wherein the ligand is biotin and the receptor specific for the ligand is avidin or streptavidin.   The method for profiling protein kinase activity according to any one of claims 1 to 6, wherein the chelate compound is a polyamine zinc complex.   The method for profiling protein kinase activity according to any one of claims 1 to 7, wherein a dinuclear zinc complex having a polyamine compound as a ligand is used as the chelate compound. 9. The protein kinase activity profiling method according to any one of claims 1 to 8, wherein a compound represented by the formula (I) is used as a chelate compound.
  The method for profiling protein kinase activity according to any one of claims 1 to 9, wherein the peptide is immobilized via a thiol group.   The method for profiling protein kinase activity according to any one of claims 1 to 10, wherein the number of peptides to be immobilized is 24 or more.   The method for profiling protein kinase activity according to any one of claims 1 to 11, wherein the number of peptides to be immobilized is 48 or more.   The method for profiling protein kinase activity according to any one of claims 1 to 12, wherein the number of peptides to be immobilized is 96 or more.