JP2007282519A - Peptide array for detecting phosphorylation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analysis method by which highly reliable data can be obtained, when a bonding signal is amplified to especially detect phosphorylation with a protein kinase used for a surface plasmon (SPR) measurement in a measurement system using a peptide array without receiving the non-specific affections of biological molecules. <P>SOLUTION: This peptide array for detecting the phosphorylation of an immobilized peptide is characterized in that the peptide whose one terminal is a cysteine residue and into whose amino acid sequence a hydrophilic compound such as PEG is inserted is immobilized on a substrate through a cross-linking agent having a mol.wt. of ≤500, and a phosphorylation detection method using the peptide array. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for detecting phosphorylation on a chip by using, for example, surface plasmon resonance (SPR). More specifically, in a method for detecting phosphorylation of an immobilized peptide by allowing a solution containing or thought to contain a protein kinase to act on a chip on which the peptide is immobilized, polyethylene glycol ( Detection technology characterized by using a chelate compound in the detection of phosphorylation using an array in which a peptide with a hydrophilic polymer such as (PEG) is immobilized via a low molecular weight cross-linking agent About.

  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. In particular, in the post-genome, post-translational modification of proteins, especially analysis of phosphorylation, is important for elucidating in detail the regulation of protein activity and function.

  As a related technique that has already been reported, for example, a technique in which a peptide is directly synthesized on a cellulose membrane by the SPOT technique and then immobilized on a chip is known. It has been reported that a p60 tyrosine kinase substrate was immobilized on a chip using this technique, and kinase activity was evaluated using a fluorescent substance or a radioactive substance (see, for example, Non-Patent Document 1). In addition, the activity of protein kinase A (hereinafter sometimes referred to as PKA) and NIMA-related kinase 6 (NEK6) was assayed using a radioactive substance using a peptide array prepared by the same technique. There is also a report about this (for example, see Non-Patent Documents 2 and 3). In addition, there is an example in which a biotin-bonded peptide is immobilized on a substrate coated with avidin and PKA activity is examined (for example, see Non-Patent Document 4). However, in any of the above methods, the background at the time of detection has an adverse effect on observing the spot, and its reduction is a major issue. Furthermore, the method using radioactive materials is the mainstream, and there is a problem that it is necessary to install a dedicated facility as well as safety, and it can be carried out only by a limited research institution. Alternatively, in the case of a method using an antibody, there are problems such as insufficient cost and required characteristics, and it is difficult to say that the method is always satisfactory.

Curr. Opin. Biotechnol. 13,315,2002 Angew. Chem. Int. Ed. 43,2671,2004 Nature Methods 1,27,2004 J. et al. Biol. Chem. 277, 27839, 2002

  An object of the present invention is to provide an evaluation system for detecting protein kinase activity in an on-chip that is capable of high throughput for drug discovery screening by a simple and safe method.

（６）アレイ表面が金であることを特徴とする（１）〜（５）のいずれかのペプチドアレイ。
（７）基板が透明基板であることを特徴とする（１）〜（６）のいずれかのペプチドアレイ。
（８）（１）〜（７）のいずれかのペプチドアレイ上におけるリン酸化を、キレート化合物を用いて検出することを特徴とするリン酸化の検出方法。
（９）キレート化合物がポリアミン亜鉛錯体であることを特徴とする（８）のリン酸化の検出方法。
（１０）キレート性化合物がポリアミン化合物を配位子として有する二核亜鉛錯体であることを特徴とする（８）または（９）のリン酸化の検出方法。
（１１）キレート性化合物が式（ＩＩＩ）に記載される構造を有する化合物であることを特徴とする（８）〜（１０）のいずれかのリン酸化の検出方法。

（１２）アレイが表面プラズモン共鳴（ＳＰＲ）解析に用いられることを特徴とする（８）〜（１１）のいずれかのリン酸化の検出方法。 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. That is, the present invention has the following configuration.
(1) An array for detecting phosphorylation of an immobilized peptide, wherein one end of the peptide is a cysteine residue and a hydrophilic compound is inserted into the amino acid sequence. A peptide array which is immobilized on a substrate via a cross-linking agent having a molecular weight of 500 or less.
(2) The peptide array of (1), wherein the hydrophilic compound has a molecular weight of 100 to 1,000.
(3) The peptide array according to (1) or (2), wherein the hydrophilic compound has a molecular weight of 400 to 1,000.
(4) The peptide array according to any one of (1) to (3), wherein the hydrophilic compound is polyethylene glycol (PEG).
(5) The peptide array according to any one of (1) to (4), wherein the crosslinking agent is a compound represented by the formula (I) or the formula (II).

(6) The peptide array according to any one of (1) to (5), wherein the array surface is gold.
(7) The peptide array according to any one of (1) to (6), wherein the substrate is a transparent substrate.
(8) A method for detecting phosphorylation, which comprises detecting phosphorylation on the peptide array according to any one of (1) to (7) using a chelate compound.
(9) The method for detecting phosphorylation according to (8), wherein the chelate compound is a polyamine zinc complex.
(10) The method for detecting phosphorylation according to (8) or (9), wherein the chelating compound is a dinuclear zinc complex having a polyamine compound as a ligand.
(11) The method for detecting phosphorylation according to any one of (8) to (10), wherein the chelating compound is a compound having a structure described in formula (III).

(12) The method for detecting phosphorylation according to any one of (8) to (11), wherein the array is used for surface plasmon resonance (SPR) analysis.

A measurement system for detecting protein kinase activity on-chip can be obtained by the interaction analysis using the array of the present invention, particularly by SPR. It is also useful to be able to cope with high throughput by performing analysis with an array. In particular, the pharmaceutical industry is expected to be very useful as an evaluation system for novel drug discovery screening such as phosphorylation inhibitors. The peptide array of the present invention can obtain an excellent phosphorylation signal because the flexibility of the immobilized peptide is simultaneously given without reducing the yield of the immobilized stabilization reaction.
The peptide array of the present invention can obtain an excellent phosphorylation signal because the flexibility of the immobilized peptide is simultaneously given without reducing the yield of the immobilized stabilization reaction.

  The present invention is characterized by comprehensive detection of phosphorylation by protein kinase in an on-chip by, for example, interaction analysis by SPR. In particular, the use of SPR has the advantage of being able to perform label-free analysis. Compared to the need for detection by means of labeling with a radioisotope or the like in general chips and arrays, not only convenience but also safety. This is also an advantage. In this case, it is possible to detect only whether or not the substance is finally bound, and even if a substance not related to the interaction is adsorbed non-specifically, it is not erroneously detected. Therefore, if the interacting target substance is not non-specifically adsorbed to the negative control, it can be determined that the measurement is accurate.

  The material of the substrate used in the present invention is not particularly limited, but metals such as gold and silver are preferably used. Of these, gold which is very stable against acids, alkalis, organic solvents and the like is preferable. 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. A 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 metal thin 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, which applies this SPR to array analysis technology, irradiates polarized light beams over a wide area and analyzes the reflected image to monitor the interaction between substances by making full use of image processing technology. 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, light 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 a pinhole is preferable as a means for obtaining a light beam having a uniform brightness, but there is a drawback that the illuminance decreases when 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 condensed to pass 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 a half-value width, but the smaller the half-value width, the sharper the wavelength distribution is, and more specifically, the half-value width is preferably 100 nm or less. An image photographed by the CCD camera through the optical interference filter is taken into a computer, and a change in brightness of a certain part can be evaluated in real time, and an entire 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 purpose of evaluating the reflection image from the metal thin film, the SPR resonance angle is as small as possible, and the captured image is not likely to be distorted and is easy to analyze. 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.

  In the present invention, an array in which peptides are immobilized on a substrate as described above is used. As the peptide, at least one peptide containing an amino acid sequence capable of functioning as a protein kinase substrate, preferably a plurality of amino acid sequences that are phosphorylated by different types of protein kinases, is used. Furthermore, in order to confirm the reliability regarding the measurement, it is preferable that a pre-phosphorylated amino acid residue (positive control) or a negative control is also immobilized on the same substrate. When a negative control is used, it is preferable that the phosphorylation site is a serine residue, a threonine residue is substituted with an alanine residue, and a phosphorylation site is substituted with a phenylalanine residue when the phosphorylation site is a tyrosine residue. In terms of ease of synthesis, ease of handling, storage stability, etc., it is preferable to use a peptide having a relatively low molecular weight. Here, the peptide refers to a commonly used meaning, in which two or more amino acids are linked by peptide bonds. The number of amino acid residues is not particularly limited, but is usually about 5 to 60 residues, preferably about 7 to 30 residues, and more preferably about 10 to 25 residues.

  The method for immobilizing peptides in the peptide array of the present invention is characterized by immobilizing via a thiol group in a cysteine residue added to the end of the peptide. It is particularly preferable from the viewpoints of specificity and sensitivity.

  It is preferable that 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. When a cysteine residue is added to one end, only a cysteine residue may be added, but a spacer is used in order to increase the efficiency of interaction with a substance that acts by increasing the degree of freedom of the immobilized peptide. As an amino acid sequence of 1 to several residues may be further added. The amino acid sequence of the spacer portion is not particularly limited, but it is particularly preferable to add a sequence having 1 to several glycine residues and / or alanine residues or serine residues.

  The present invention is characterized in that a hydrophilic compound is inserted 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, 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 less likely 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. Preferably it comprises two or more times.

  Here, the hydrophilic compound means a compound having a repeating unit having a property soluble in water or swelled in water, and may be a synthetic product or a natural product. Specifically, as the hydrophilic polymer, for example, polyethylene glycol, polyvinyl alcohol, poly (meth) acrylic acid, poly (meth) acrylate, poly (meth) acrylamide, polyethyleneimine, polyvinylpyrrolidone, carboxylic acid Alternatively, there may be mentioned polyesters, polyurethanes, carboxymethylcellulose, and polysaccharides such as chitosan, carrageenan, and glucomannan, which are copolymerized with a salt thereof, a monomer containing sulfonic acid or a salt thereof, or a hydrophilic moiety such as polyethylene glycol. Of these, polyethylene glycol (PEG) is particularly preferable. By adding such a spacer, the degree of freedom of the immobilized substrate peptide can be improved, and as a result, the access to the immobilized peptide of protein kinase is facilitated, so that the efficiency of the phosphorylation effect received is improved. It becomes possible to raise more.

  Moreover, you may use the state in which the compound which has a thiol group is chemically combined in the amino acid residue in one or more places with respect to the peptide fix | immobilized. The bonding position of the compound is not particularly limited, but is preferably bonded to any terminal amino acid residue. Further, the position at which the hydrophilic compound is inserted is not particularly limited as long as it is between the immobilization site and the site subjected to phosphorylation, but it is more close to the immobilization site. preferable. The method for synthesizing the peptide into which the hydrophilic compound is inserted is not particularly limited, and can be easily obtained by using, for example, a commercially available compound modified with a protecting group for peptide synthesis. The kind of the protecting group is not particularly limited, and general groups such as an Fmoc group and a tBoc group are used.

  In the present invention, when a peptide is immobilized via a thiol group, a heterobifunctional cross-linking agent having a succinimide (NHS) group or a sulfated succinimide group and a maleimide (MAL) group is introduced after introducing an amino group to the surface in advance. It is particularly preferable to use it. As such a heterobifunctional cross-linking agent, it is possible to use a hydrophilic polymer such as PEG in which both ends are modified with NHS groups and MAL groups, but the immobilization yield of the peptide is improved. In order to achieve this, a lower molecular weight is better used. The molecular weight is 500 or less, preferably 200 to 500, and more preferably 300 to 400. Specifically, the compound Succinimidyl 4- [N-maleimidomethyl] 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (hereinafter referred to as SMCC) or the compound Sulfosuccinimidyl 4- [N-maleimidocylimide] represented by the formula (II) -1-carboxylate (hereinafter referred to as SSMCC). It should be noted that not only compounds having the same structure as the compound represented by formula (I) or formula (II) but also compounds analogized within a range not impairing the function thereof are included. In the present invention, both SMCC and SSMCC can be applied. However, 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. Both can be used properly depending on the solubility of the peptide.

  By applying low molecular weight compounds as described above, the efficiency of immobilizing peptides on the chip is significantly higher than when high molecular weight substances are used as cross-linking agents. As a result, the signal due to the binding becomes clearer and the flexibility of the immobilized peptide is provided. Further, there is almost no problem with non-specific influences, which is advantageous in that the so-called S / N ratio can be increased. However, the type of the crosslinking agent in the present invention is not particularly limited to these.

  In order to introduce the SMCC or SSMCC onto a chip to form a maleimide surface, a succinimide group at the other end of the SMCC or a functional group reactive with the succinimide sulfate group at the other end of the SSMCC, Specifically, it is necessary to introduce an amino group on the chip in advance. 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.

  In the peptide array used in the present invention, the non-immobilized portion (background portion) of the peptide is preferably coated with a compound as shown in formula (IV). In formula (IV), m represents an integer of 1 to 20, and n represents an integer of 1 to 10. The value of m is more preferably in the range of 2 to 10, and still more preferably in the range of 4 to 8. As for the value of n, the range of 2-8 is more preferable, and the range of 3-6 is still more preferable. By coating the background portion with this compound, non-specific adsorption in the background portion can be very effectively suppressed. In addition, the peptide array of the present invention improves the reproducibility of data depending on various fluctuation factors such as the substrate production lot and processing conditions, and can obtain very stable measurement data.

  However, 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 the nonspecific adsorption more severely is required, the above 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 surface of the unreacted maleimide group, a compound having a thiol group is preferably used, and a PEG (polyethylene glycol) derivative is particularly preferably used.

The present invention is directed to the detection of phosphorylation on the array. Therefore, when a solution containing or considered to contain protein kinase is allowed to act on the array obtained as described above, a compound that inhibits or is thought to inhibit the protein kinase activity is coexisted in the solution. I will let you. The protein kinase to be subjected to profiling may be a commercially available reagent, but it is also possible to use a protein kinase already contained in or estimated to be contained in a cell-derived extract. For example, by immobilizing a protein kinase reagent or nucleoside triphosphate (ATP), which is already contained in a buffer or cell extract or a mixture of both, and acting on the array directly, the immobilized substrate Can be phosphorylated. The phosphorylation conditions vary depending on the type of protein kinase, but it is usually about 10 to 40 ° C., preferably about 20 to 40 ° C. for about 5 minutes to 8 hours, preferably about 10 minutes to 5 hours. Thus, the immobilized peptide can be phosphorylated. Moreover, you may coexist the substance which assists and accelerates | stimulates phosphorylation, such as cAMP, cGMP, Mg ^<2+> , Ca ^{< 2+} >, in a reaction liquid as needed.

  The present invention can also be aimed at detecting phosphorylation inhibitory activity on the array. In this case, when a solution containing or thought to contain a protein kinase is allowed to act, a compound that inhibits or is thought to inhibit the protein kinase activity coexists in the solution. The coexisting inhibitor is not particularly limited, but may be a peptide or protein, or other low molecular weight compound. Low molecular compounds include those having a molecular weight of 2,000 or less, but are not particularly limited.

  By the way, when a peptide is phosphorylated, the molecular weight is only increased by 80, so that when a detection by SPR is attempted, it is difficult to obtain a direct detection signal. Therefore, it is necessary to use a substance or compound that can specifically bind to a phosphate group or a phosphorylated amino acid as a detection probe. In the present invention, a chelate compound is used as the type of detection probe, in particular for specifically monitoring 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) as shown in the formula (III). A binuclear zinc complex having a polyamine compound having [bis (2-pyridinemethyl) amino] -2-propanolatodiincinc (II) complex) as a ligand (provided that the hydroxyl group of the propanol skeleton is divalent to two zinc atoms as an alcoholate) 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) can be 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 chemical structure can be confirmed by elemental analysis, nuclear magnetic resonance analysis, and infrared analysis. Examples of the data are shown below.
Theoretical value of elemental analysis _{··· C 29 H 34 Cl 2 N} 6 O 12 Zn 2: C, 40.49; H, 3.98; N, 9.77
Measured value of elemental analysis: C, 40.43; H, 3.86; N, 9.85
¹ H NMR (500 MHz, DMSO-d ₆ ) result δ 2.04 (2H, dd, J = 12.1 and 12.3 Hz, CCHN), 2.53 (3H, s, CH ₃ ), 3.06 (2H, dd, J = 12.1 and 12.3 Hz, CCHN), 3.74 (1H, t, J = 10.4 Hz, CCHC), 4.02-4.34 (8H, m, ArCH ₂ ), 7.54-7.65 (8H, m, ArH), 8.06-8.12 (4H, m, ArH), 8.58 (4H, m, ArH) ¹³ C NMR (125 MHz, DMSO) -D ₆ ) result δ 58.0, 60.1, 62.0, 64.6, 122.7, 124.3, 124.4, 124.4, 139.9, 140.4, 147.0, 147.2, 154.7, 1 5.1
Infrared analysis results 1609, 1576, 1556 (C = O), 1485, 1439, 1266, 1108 (ClO ^4- ), 1090 (ClO ^4- ), 770, 625 cm ^-1
The above data show that the compound of formula (I) has 1 equivalent of acetate ion and 2 equivalents of perchlorate ion as counter ions.

  In addition, although the solution density | concentration of the chelate compound which acts in this invention is not specifically limited, Usually, it is preferable to set it as the range of 0.001-10M, especially 0.01-1M.

    Furthermore, for example, Anal. Chem. Vol. 77, pp. As reported in 3979-3985 (2005), a biotin-modified zinc chelate compound (see formula (V)) is used to act streptavidin, and further anti-streptavidin antibodies, A method of specifically amplifying the SPR signal resulting from phosphorylation is also preferred. In this case, the solution concentration of the biotin-modified polyamine zinc complex 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, 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 action temperature may be room temperature or may be incubated at a constant temperature of 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 is also possible to use fluorescence or luminescence as the detection means. In this case, it is preferable to act a receptor such as avidin or streptavidin after the biotin-modified polyamine zinc complex is allowed to act. 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 that case, avidin or streptavidin labeled with a fluorescent substance or a chemiluminescent substance as described above may be used.

  Alternatively, after the 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 increased. In that case, it is preferable to use those labeled with a fluorescent substance or a chemiluminescent substance as described above. 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 more preferable 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.

  In this case, a fluorescent substance or a chemiluminescent substance that is well known in the art is used. The fluorescent substance is not particularly limited, and any fluorescent compound is applied. For example, FITC, 6-FAM. Examples thereof include those used in general fluorescence assays such as Cy3, Cy5, Texas Red, TAMRA, and APC. Moreover, it is not specifically limited about the case where a chemiluminescent substance is used, For example, common compounds, such as a luminol, a luciferin, and a lophine, are illustrated.

  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 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. And has a huge 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.

  By conducting the phosphorylation reaction on-chip by the method of the present invention as described above, comprehensive analysis by profiling of protein kinase activity can be realized. In particular, by monitoring the phosphorylation inhibitory activity, it is possible to provide a technique useful for drug discovery screening.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not particularly limited to the examples.

[Example 1]
(Preparation of SPR array)
As shown in the formula (VI), a linear thiol PEG reagent (SPSPT-0011 manufactured by SensoPath) whose terminal functional group is a thiol group was dissolved in 7 ml of ethanol at a concentration of 1 mM. The molecular weight of the linear thiol PEG is 336.54. In particular, it shows metal bondability 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 linear PEG thiol solution for 3 hours to bond PEG 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 PEG 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. PEG remains in the unirradiated portion, and functions as a reference portion as a background portion 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 20 mM phosphate buffer (150 mM NaCl; pH 7.2) at 0.4 mg / ml and reacted with 8-AOT on the gold surface for 15 minutes. Since the amino group of 8-AOT and the NHS group of SSMCC reacted and the MAL group remained unreacted, a maleimide group could be introduced to the surface via PEG.

  On the surface obtained as described above, a peptide (tyrosine type; SEQ ID NO: 1) consisting of an amino acid sequence serving as a substrate for cSrc kinase, its negative control (phenylalanine type; SEQ ID NO: 2), positive control (phosphorylated tyrosine type) SEQ ID NO: 3) and a peptide in which PEGs having different lengths were inserted were each dissolved in a phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 1 mg / ml, and MultiSPRinter (registered trademark) Spotting was performed 10 nl at a time using an automatic spotter (manufactured by Toyobo). PEG insertion was carried out using N-Fmoc-amino-dPEG-acid manufactured by Quanta Biodesign as the Fmoc-modified PEG derivative at the time of peptide synthesis, and the PEG chain length (number of single-chain repeats) was 4, 8, 12 Can be obtained using 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) SUNBRIGHT (registered trademark) MESH-50H manufactured by NOF Corporation is dissolved in a phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) to a concentration of 1 mM, and 250 μl is poured onto the chip at room temperature. The reaction was carried out for 1 hour. The molecular weight of PEG thiol used here is 5,000.

[Example 2]
(Detection of phosphorylation inhibitory activity by cSrc kinase by SPR analysis)
Phosphorylation with cSrc was performed on the array that had been blocked as described above. 300 μl of cSrc kinase solution was dropped on the array and reacted at 30 ° C. for 30 or 60 minutes. The composition of the cSrc solution was 6 μl of cSrc kinase (Upstate; 5 U / μl), 274 μl of 50 mM MES buffer (pH 6.8), 15 μl of 1M magnesium chloride solution, and 3 μl of 1 mM ATP.

  The array subjected to the above treatment was washed with PBS and water, and a biotin-modified polyamine zinc complex was allowed to act on the array. As the biotin-modified polyamine zinc complex, Phos-tag (registered trademark) BTL-104 (Nard Laboratories, Inc.) represented by the following formula (V) was used. Phos-tag (registered trademark) BTL-104 has a concentration of 2 μg / ml, and the lysis solution contains 10 mM HEPES- containing 0.005% Tween 20, 10% (v / v) ethanol, 0.2 M sodium nitrate, 1 mM zinc nitrate. NaOH buffer (pH 7.4) was used. Incubation was for 30 minutes at room temperature.

  Thereafter, the array was washed three times with PBS and water, and the surface of the array was dried. Then, the array was set in an SPR imaging device (MultiSPRinter (registered trademark): manufactured by Toyobo Co., Ltd.), and 50 mM Tris-HCl buffer ( pH 7.4) was flowed into the flow cell at a rate of 100 μl / min. After confirming that the signal from the SPR was stabilized, a streptavidin (Molecular Probes) solution having a concentration of 1 μg / ml was injected into the cell in the SPR device to act. The change in the SPR signal at that time was observed, and when the signal rise reached a plateau state, the running buffer was sent again to perform washing, and the anti-streptavidin antibody was similarly allowed to act at a concentration of 1 μg / ml. . Also in this case, when the SPR signal rise reached a plateau state, the running buffer was fed again for washing. 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. In the SPR analysis, an image is captured every 5 seconds by a CCD camera, and from the image captured at the time after the antibody reaction, the image at the time before the reaction is imaged image processing software Scion Image (manufactured by Scion Corp.). It is the result of having performed the subtraction process using.

  As shown in FIG. 1, comparing the substrate without PEG (No. 7) and the substrate with PEG inserted (No. 4, 5, 6), the latter is much stronger. In addition, a reasonable phosphorylation signal is shown in comparison with the positive control (No. 2). In the negative control (No. 1) and the blank in which the peptide is not immobilized (No. 3), almost no signal is confirmed. From this result, it was considered that the detection sensitivity of on-chip phosphorylation was effectively improved by the insertion of PEG. This reflects the result that the spacer effect by PEG insertion has improved the degree of freedom of the immobilized peptide, which facilitated access to the immobilized substrate peptide of cSrc kinase and increased phosphorylation efficiency. it is conceivable that.

[Example 3]
In the same manner as in Example 1, a maleimide group was introduced onto the gold chip surface via PEG, and then the cSrc substrate having the arrangement shown in FIG. In the same manner as in Example 1, an array was prepared by spotting and immobilizing the cSrc substrate peptide into which the peptide and PEG (repeated 12 times of single chain) were inserted. After blocking in the same manner as in Example 1, phosphorylation with cSrc was performed for 2 hours and 5 hours under the kinase concentration condition of 1/2 that in Example 2. The total composition of the cSrc solution in this case was 300 μl, 3 μl of cSrc kinase (Upstate; 5 U / μl), 277 μl of 50 mM MES buffer (pH 6.8), 15 μl of 1M magnesium chloride solution, and 3 μl of 1 mM ATP. Thereafter, the results of SPR imaging performed in the same manner as in Example 2 are shown in FIG.

  When the cSrc concentration was lowered, almost no signal change was observed in the substrate peptide (No. 1) in which PEG was not inserted, whereas in the substrate (No. 4) in which PEG was inserted, It was confirmed that the signal increased. This result also confirmed that the phosphorylation efficiency by PEG insertion was effectively increased.

  By utilizing the present invention, it is possible to comprehensively analyze many types of protein kinase signals, and to effectively profile protein kinase dynamics in cells accompanying the introduction of genes with unknown functions or drug administration. it can. This is expected to expand into genomic drug discovery, such as functional analysis from new genes and approaches to drug discovery.

In Example 2, it is a figure which shows the result of having observed the mode of phosphorylation in On-chip of the fixed peptide by cSrc by SPR imaging. In Example 3, it is a figure which shows the fixed arrangement | positioning of the substrate peptide in the produced array. In Example 3, it is a figure which shows the result of having observed the mode of phosphorylation in On-chip of the fixed peptide by cSrc by SPR imaging.

Claims (12)

  An array for detecting phosphorylation of an immobilized peptide, wherein one end of the peptide is a cysteine residue, and a peptide having a hydrophilic compound inserted into the amino acid sequence has a molecular weight of 500 A peptide array, which is immobilized on a substrate via the following cross-linking agent.   The peptide array according to claim 1, wherein the hydrophilic compound has a molecular weight of 100 to 1,000.   The peptide array according to claim 1 or 2, wherein the hydrophilic compound has a molecular weight of 400 to 1,000.   The peptide array according to any one of claims 1 to 3, wherein the hydrophilic compound is polyethylene glycol (PEG). The peptide array according to any one of claims 1 to 4, wherein the crosslinking agent is a compound represented by the formula (I) or the formula (II).

  The peptide array according to any one of claims 1 to 5, wherein the array surface is gold.   The peptide array according to claim 1, wherein the substrate is a transparent substrate.   The phosphorylation detection method characterized by detecting phosphorylation on the peptide array in any one of Claims 1-7 using a chelate compound.   The method for detecting phosphorylation according to claim 8, wherein the chelate compound is a polyamine zinc complex.   The method for detecting phosphorylation according to claim 8 or 9, wherein the chelating compound is a dinuclear zinc complex having a polyamine compound as a ligand. The method for detecting phosphorylation according to any one of claims 8 to 10, wherein the chelating compound is a compound having a structure described in formula (III).

  12. The method for detecting phosphorylation according to claim 8, wherein the array is used for surface plasmon resonance (SPR) analysis.

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