JP2009050171A - Method for detecting phosphorylation on substrate by surface plasmon resonance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an evaluation system for exhaustive on-chip analysis of protein kinase activities, enabling throughput by a simple and safe method. <P>SOLUTION: The peptide array is obtained by fixing a plurality (five or more kinds) of peptides being substrates of a protein kinase and having nine to eleven specific amino acid sequences on a gold surface, and is used for detecting the phosphorylation of the peptide by the kinase by a surface plasmon resonance imaging after allowing an antibody or a metal complex compound capable of recognizing a phosphorylation site to act thereon. The method for detecting the phosphorylation uses the peptide array. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for detecting phosphorylation on a chip by using surface plasmon resonance (SPR). More specifically, the present invention relates to a method for detecting phosphorylation of an immobilized peptide substrate by allowing a solution containing or thought to contain a protein kinase to act on a chip on which the peptide substrate is immobilized.

  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. A technique for comprehensively analyzing the activity of protein kinases responsible for phosphorylation is required. As a means for that, array analysis is advantageous in terms of operability and cost, but a sufficient one has not been established yet. Furthermore, in order to comprehensively analyze protein kinase activity, selection of various protein kinase substrates is very important.

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, using a peptide array produced by the same technique, activities such as protein kinase A (hereinafter sometimes referred to as PKA) and NIMA-related kinase 6 (NEK6) were assayed using radioactive substances. 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 comprehensive analysis of protein kinase activity in an on-chip, which is capable of high-throughput for drug discovery screening by a simple and safe method. .

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) Phosphorylation on a substrate, characterized in that at least two or more peptide substrates containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-26 are immobilized on a gold surface Peptide array for detecting phosphorylation by resonance.
(2) The peptide array of (1) for detecting phosphorylation on a substrate by surface plasmon resonance imaging.
(3) The peptide array according to (1) or (2), wherein 5 or more types of peptide substrates containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 are immobilized.
(4) The peptide array according to any one of (1) to (3), wherein 10 or more peptide substrates containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 are immobilized.
(5) The peptide array according to any one of (1) to (4), wherein the peptide substrate containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 has a cysteine residue at the terminal .
(6) A peptide substrate containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 is immobilized on a gold surface using a compound represented by formula (I) or (II) as a cross-linking agent. The peptide array according to any one of (1) to (5).

(7) Surface plasmon obtained by allowing an antibody capable of recognizing a phosphorylated site to act after phosphorylating on a substrate using the peptide array of any one of (1) to (6) A method for detecting phosphorylation by a resonance signal.
(8) Using the peptide array of any one of (1) to (6), the phosphorylation reaction is performed on the substrate, and then a metal complex compound capable of recognizing the phosphorylation site is allowed to act. A method for detecting phosphorylation by a signal of surface plasmon resonance.
(9) The method for detecting phosphorylation according to (8), wherein the metal complex compound is modified with biotin, and the metal complex compound is allowed to act on the peptide array and then avidin or streptavidin is allowed to act. .
(10) The method for detecting phosphorylation of (8) or (9), wherein the metal complex compound is a zinc chelate compound.
(11) The method for detecting phosphorylation according to any one of (8) to (10), wherein the metal complex compound is a compound represented by the formula (III).

(12) The method for detecting phosphorylation according to any one of (1) to (11), wherein a phosphorylation reaction on a substrate by cell lysate is detected.
(13) The method for detecting phosphorylation according to (12), wherein a phosphorylation reaction on a substrate by a lysate of a cell stimulated with a drug is detected.

  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. It is also very useful for SPR measurement using cell lysate. In particular, it is expected to be very useful as an evaluation system for diagnosis of diseases such as cancer and new drug discovery screening such as phosphorylation inhibitors.

  The present invention is characterized by comprehensive detection of phosphorylation by protein kinase in an on-chip by interaction analysis by SPR. 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, etc., in general chips and arrays, it is not only simple but also safe. But it is 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 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, which applies this SPR to array analysis technology, monitors 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, etc. 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, a means for irradiating the chip with a polarized light beam in a wide range and analyzing the reflected image and sufficiently securing the illuminance of the light beam is necessary. 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 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, and more preferably 1.6 or more.

  In the present invention, an array in which a peptide substrate is immobilized on a substrate as described above is used. The peptide substrate contains at least 2 peptides, preferably 5 or more, more preferably peptides containing the amino acid sequences of SEQ ID NOs: 1 to 26 as those containing amino acid sequences that can function as substrates for various protein kinases. Uses 10 or more, more preferably 20 or more. These peptides have been confirmed to be responsive to various protein kinases. By selecting some of these 26 types as appropriate and using them in combination for array analysis, comprehensive protein kinase activity can be obtained. Can be analyzed. As a detection means in array analysis, the detection method by SPR is particularly advantageous from the viewpoint of quantitative reproducibility, in addition to a method capable of measurement without labeling, for example, compared to a method such as fluorescence array analysis.

  The 26 peptides and corresponding kinases are shown in Table 1 below.

  As shown in Table 1 above, since 26 peptides are substrates recognized by specific kinases, by combining them, one or more kinase activities can be easily and simultaneously detected. .

  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 a peptide substrate in the peptide array of the present invention is not particularly limited, and is a method via a functional group such as an amino group or a thiol group in a peptide sequence, such as an affinity such as a His tag, GST tag, or MBP tag. For example, a method using a bond. Among these, a method of immobilizing a peptide substrate via a thiol group is particularly preferable from the viewpoints of both specificity and sensitivity.

  When immobilized via a thiol group, it is preferable that at least one cysteine residue is present in the amino acid sequence of the peptide. 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 substrate to be immobilized is not particularly limited, but preferably at least in the peptide portion (which is surrounded by amino acids at both ends and may have a spacer between the amino acids). It is better to add one end, more preferably only 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, 100-1,000 are preferable and 400-1,000 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. 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 Or the salt, the sulfonic acid, the monomer containing the salt, or polyester, polyurethane, carboxymethylcellulose which copolymerized hydrophilic parts, such as polyethyleneglycol, and polysaccharides, such as chitosan, carrageenan, and glucomannan, are mentioned. Of these, polyethylene glycol (PEG) is particularly preferable. By adding such a spacer, the degree of freedom of the immobilized peptide substrate can be improved, and as a result, the protein kinase can be easily accessed to the immobilized peptide substrate, resulting in the efficiency of the phosphorylation effect received. Can be further increased.

  Alternatively, a compound in which a compound having a thiol group is chemically bonded at any one or more amino acid residues to the peptide substrate to be immobilized may be used. 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 substrate is immobilized via a thiol group, a heterobifunctional cross-linking agent having a succinimide (NHS) group or a succinimide sulfate group and a maleimide (MAL) group after previously introducing an amino group onto the surface It is particularly preferable to use 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. In order to improve, a lower molecular weight may be used. 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 a low molecular weight compound as described above, the efficiency of immobilizing the peptide substrate on the chip is significantly higher than when a high molecular weight substance is used as a cross-linking agent. The effect is that the signal due to the binding becomes clearer. 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 the 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 substrate is preferably coated with a compound as shown in the 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 disruption solution. For example, the protein kinase reagent or nucleoside triphosphate (ATP) added to the protein kinase reagent or cell disruption solution in the buffer or cell disruption solution (cell lysate) or a mixture of both is directly applied to the array. By this, phosphorylation of the immobilized substrate can be performed.

  In particular, by applying a cell lysate that has been stimulated with various drugs to the present invention, it is possible to capture changes in response patterns of various protein kinase activities by the drugs. It is also possible to evaluate phosphorylation inhibition in a system in which an inhibitor coexists. Therefore, for example, screening for novel drugs such as inhibitors of various protein kinases, diagnosis of diseases such as cancer, etc. using protein kinase as a marker is also possible.

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 particular, an antibody that recognizes a phosphorylated protein can be used as a type of detection probe for specifically monitoring the phosphorylation of a substrate on the array with high sensitivity. Specifically, phosphorylated serine, phosphorylated threonine, or phosphorylated tyrosine antibody is used. As the antibody, either a polyclonal antibody or a monoclonal antibody can be applied, but a monoclonal antibody is generally preferable in terms of specificity.

  A method using a chelate compound can also be applied as the detection probe. 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 (V). 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) The present invention is not particularly limited to this compound (see Dalton Trans Vol. 8, pp. 1189-1193 (2004)).

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 (V) 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 solution of 1,3-bis [bis (2-pyridylmethyl) amino] -2-hydroxypropane in ethanol is added 10M aqueous sodium hydroxide, followed by zinc acetate dihydrate. A brown oil can be obtained by distilling off the solvent under reduced pressure. After adding water to this residue and dissolving, 1M sodium perchlorate aqueous solution was added dropwise while heating to 70 ° C., and the precipitated colorless crystals were collected by filtration and dried by heating to form the structure of formula (V). two perchlorate ^{_{(Zn 2 L-CH 3 COO}} - · 2ClO 4 - · H 2 O) of the acetate ion adduct of the formula can be obtained in high yield. This crystal contains one molecule of crystal water.

  Furthermore, for example, Anal. Chem. Vol. 77, pp. As reported in 3979-3985 (2005), a biotin-modified zinc chelate compound (see formula (III)) is used to act avidin or streptavidin, and even anti-streptavidin antibodies A method of specifically amplifying an 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.

  As described above, after the biotin-modified zinc chelate compound is allowed to act, avidin or streptavidin is allowed to act, and further, an antibody that recognizes avidin or streptavidin is allowed to act, thereby further enhancing detection sensitivity. Become. In this case, the concentration when avidin or streptavidin or an 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.

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

  The application of such a chelating compound is advantageous in that it can be synthesized at a very low cost by the method described above. In addition, it is stable and easy to use in that it can be stored at room temperature, which is advantageous in terms of distribution. Compared 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, it is expected to be a useful technique in the fields of drug discovery screening and cancer diagnosis.

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, the peptide consisting of the amino acid sequence represented by SEQ ID NOs: 1 to 26 was formed in a phosphate buffer solution (20 mM phosphate, 150 mM NaCl; pH 7.2) was dissolved at a concentration of 0.5 mM and spotted by 10 nl using a MultiSPRinter (registered trademark) automatic spotter (manufactured by Toyobo). All peptides used were those with a cysteine (Cys) -glycine (Gly) -glycine (Gly) sequence added to the end. 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 (wherein one functional group 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 by Src kinase by SPR analysis)
Phosphorylation with Src was performed on the chip using the array subjected to blocking as described above. 300 μl of Src kinase solution was dropped on the array, and the reaction was performed at 37 ° C. for 2 hours. The composition of the Src solution was 6 μl of Src kinase (carna bioscience; 20 pg / μl), 252 μl, 15 mM Tris-HCl buffer (pH 7.5), 10 μm ATP 3 μl, 0.1% Tween 20 30 μl, 100 mM DTT 6 μl, 500 mM chloride. Magnesium was 3 μl.

  The array subjected to the phosphorylation reaction was washed with PBS and water, and after the surface of the array was dried, it was set in an SPR imaging device (MultiSPRinter (registered trademark): manufactured by Toyobo) and 50 mM Tris as a running buffer. -Hydrochloric acid buffer (pH 7.4) was flowed into the flow cell at a rate of 100 μl / min. After confirming that the signal from SPR was stabilized, a 1.3 μg / ml concentration solution of phosphorylated tyrosine antibody PT-66 (manufactured by Sigma) was injected into the cell in the SPR device to act. The change of the SPR signal at that time was observed, and when the signal rise reached a plateau state, the running buffer was again fed and washed. In addition to the spot site of each substrate, the signal change was also observed in the background.

(Observation results and discussion)
The result of SPR imaging is shown in FIG. In the SPR analysis, the image is captured every 5 seconds by the CCD camera, and the image at the time before the reaction is obtained from the image captured at the time after the antibody reaction by the image calculation processing software Scion Image (manufactured by Scion Corp.). It is the result of having performed the subtraction process using.

  Moreover, the result of having graphed the signal value in the SPR sensorgram obtained in each probe was shown in FIG. The signal value is the result of averaging the values at 3 points and subtracting the signal value at the blank. In No. 23, 9, 20, 22, 13, 5, and 19, particularly strong signals are obtained. Most of these have confirmed the usefulness of Src as a substrate in fluorescent array analysis and mass spectrum analysis separately examined.

[Example 3]
(Detection of phosphorylation by PKA by SPR analysis)
Using the blocking array obtained in the same manner as in Example 1, phosphorylation with PKA on the chip was performed. 400 μl of PKA solution was dropped on the array and reacted at 37 ° C. for 2 hours. The composition of the PKA solution was PKA (manufactured by Promega; 80 U / μl, 2.5 mg protein / ml) 1 μl, 20 mM HEPES buffer (pH 7.5) 383 μl, 10 mM ATP 8 μl, 500 mM magnesium chloride 8 μl.

  The array subjected to the phosphorylation reaction was washed with PBS and water to allow the biotin-modified polyamine zinc complex to act. As the biotin-modified polyamine zinc complex, Phos-tag (registered trademark) BTL-104 (Nard Laboratories, Inc.) represented by the following formula (III) was used. Phos-tag (registered trademark) BTL-104 has a concentration of 10 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 (as a running 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 4 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 again fed and washed, and the anti-streptavidin antibody was similarly acted at a concentration of 4 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.

  The result of SPR imaging is shown in FIG. A large difference in the imaging pattern is recognized from FIG. 2A obtained by the Src reaction. The image calculation process was performed in the same manner as in Example 2. In addition, FIG. 4 shows the results of graphing the signal values in the SPR sensorgram obtained for each probe in the same manner as in Example 2. No. In 9, 4, 16, 5, 1, 6 and 19, particularly strong signals are obtained. Most of these have been confirmed to be useful as substrates for PKA in fluorescent array analysis and mass spectrum analysis separately examined.

[Example 4]
Using the blocked array obtained in the same manner as in Example 1, detection of protein kinase activity by SPR using a cell lysate to which drug stimulation was applied was examined.
(Preparation of cell lysate)
Human breast cancer-derived MCF-7 cells (human science research resource bank) cultured in serum-free MEM were seeded in a 10 cm petri dish at 1 × 10 ⁶ cells / well and then cultured for 20 hours. Nerve growth factor (NGF) was added to the serum-free medium, added to the cells to a final concentration of 50 ng / ml, and incubated for 10 minutes in a CO ₂ incubator. After washing with 5 ml of Dulbecco's PBS (D-PBS), 1 ml of D-PBS was added, the cells were peeled off with a cell scraper, and the cell suspension was recovered. After centrifuging at 300 g for 5 minutes, the pellet was mixed with lysis buffer (50 mM potassium phosphate, pH 7.25, 1 mM EDTA, 1 mM EGTA, 10 mM magnesium chloride, 1 mM DTT, 1 mM sodium orthovanadate, 80 mM β-glycerophosphate, 3 μg / ml pepstatin. A, 5 μg / ml aprotinin, 1 mM phenylmethylsulfonylfluoride) was added, and sonication was performed 3 times for 15 seconds to disrupt the cells. The ultracentrifugation was performed 4 times at 100,000 g for 60 minutes, and the supernatant was recovered. Lysis buffer containing 2% Triton X-100 was added to the precipitate, and sonication was performed 3 times for 15 seconds to disrupt the cells. Ultracentrifugation was carried out 4 times at 100,000 g for 40 minutes, and the supernatant was collected and used. As a control, a lysate was prepared by carrying out the same treatment for cells not added with NGF. The protein contained in the lysate was quantified using a DC protein assay kit (manufactured by Bio-Rad).

(SPR analysis using cell lysate)
The cell lysate obtained above was diluted with a reaction buffer so that the protein concentration was 100 μg / ml. As final concentration conditions, 15 mM Tris-HCl (pH 7.5), 5 mM magnesium chloride, 0.01% (v / v) Tween 20, 2 mM DTT, and 0.1 mM ATP were adjusted. 300 μl of the lysate solution having the composition was dropped on the blocked array prepared in Example 1 and reacted at 37 ° C. for 2 hours. Thereafter, the array was washed for 10 minutes each while sonicating in the order of 1% SDS-PBS, 0.05% Tween 20-PBS, and milli-Q water.

  The washed array was set in an SPR imaging device (MultiSPRinter (registered trademark): manufactured by Toyobo Co., Ltd.), and SPR analysis was performed under the same method and conditions as in Example 2. FIG. 5 shows the result of graphing the signal value in the SPR sensorgram obtained for each probe. (A) is the result of measurement using cell lysate without stimulation by NGF, and (B) is the result of measurement using cell lysate stimulated by NGF. By NGF stimulation, no. In 19 substrate peptides, the increase in signal was most noticeable. In addition, no. Signals are also increasing in 22, 23, 18 and the like. Since these are substrates that showed a response in the Src reaction performed in Example 2, the results suggest the possibility that the Src activity was amplified by NGF stimulation. The most amplified signal No. Regarding 19, the response with Src was observed, but it was not particularly strong compared with other substrates. Therefore, it is speculated that activation of tyrosine kinases other than Src is also promoted.

[Example 5]
Using the lysate of NGF-stimulated cells prepared in Example 4, the response change in each probe was examined in the presence of SU6656 (Calbiochem), which is an Src inhibitor. Under the same method and conditions as in Example 4, SU-6656 was allowed to coexist at concentrations of 10 ⁻⁷ , 10 ⁻⁶ , 10 ⁻⁵ , and 10 ⁻⁴ M, and an on-chip phosphorylation reaction was performed. SPR analysis was performed in the same manner as in Example 4. The result of graphing the signal value in the SPR sensorgram obtained for each probe is shown in FIG. Regarding the peptide for which the signal is confirmed, it has been confirmed that the signal decreases as the concentration of the inhibitor increases. This also suggests that the SPR signal confirmed in Example 4 was mainly due to phosphorylation by the Src reaction.

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.

FIG. 3 is a diagram showing a pattern of immobilized peptides in the array prepared in Example 1. Numbers indicate SEQ ID NOs. It is a figure which shows the result of having performed SPR imaging obtained by Example 2 and Example 3. FIG. (A) Src reaction, (B) PKA reaction In Example 2, it is a figure which shows the SPR signal in each substrate peptide obtained by Src reaction. In Example 3, it is a figure which shows the SPR signal in each substrate peptide obtained by PKA reaction. In Example 4, it is a figure which shows the SPR signal in each substrate peptide obtained by the phosphorylation reaction by a cell lysate. (A) No NGF stimulation, (B) NGF stimulation In Example 5, it is a figure which shows the result of having examined the fluctuation | variation of the phosphorylation signal in each substrate peptide at the time of coexisting Src inhibitor SU6656 regarding the cell lysate which gave NGF stimulation.

Claims (13)

Phosphorylation on a substrate characterized by phosphorylation by surface plasmon resonance, wherein at least two or more peptide substrates containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 are immobilized on a gold surface Peptide array for detecting oxidation. The peptide array according to claim 1 for detecting phosphorylation on a substrate by surface plasmon resonance imaging. The peptide array according to claim 1 or 2, wherein five or more types of peptide substrates containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 are immobilized. The peptide array according to any one of claims 1 to 3, wherein 10 or more types of peptide substrates containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 are immobilized. The peptide array according to any one of claims 1 to 4, wherein the peptide substrate containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 has a cysteine residue at the terminal. A peptide substrate containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 is immobilized on a gold surface using a compound represented by formula (I) or (II) as a crosslinking agent The peptide array in any one of Claims 1-5.

A signal of surface plasmon resonance obtained by allowing an antibody capable of recognizing a phosphorylated site to act after phosphorylating on the substrate using the peptide array according to any one of claims 1 to 6. To detect phosphorylation. Surface plasmon resonance obtained by allowing a metal complex compound capable of recognizing a phosphorylated site to act after phosphorylating on a substrate using the peptide array according to any one of claims 1 to 6. Of detecting phosphorylation by the signal of. 9. The method for detecting phosphorylation according to claim 8, wherein the metal complex compound is modified with biotin, and after the metal complex compound is allowed to act on the peptide array, avidin or streptavidin is allowed to act. The method for detecting phosphorylation according to claim 8 or 9, wherein the metal complex compound is a zinc chelate compound. The method for detecting phosphorylation according to any one of claims 8 to 10, wherein the metal complex compound is a compound represented by the formula (III).

The method for detecting phosphorylation according to any one of claims 7 to 11, wherein a phosphorylation reaction on a substrate by cell lysate is detected. The method for detecting phosphorylation according to claim 12, wherein a phosphorylation reaction on a substrate by a lysate of a cell stimulated with a drug is detected.

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