JP4599928B2 - Peptide immobilization method - Google Patents

Description

  The present invention relates to a method for producing a peptide array used in an analysis system for detecting phosphorylation reaction. More specifically, the present invention relates to a method for producing a peptide array capable of increasing a phosphorylation signal by using a low molecular weight compound as a cross-linking agent and reducing nonspecific effects.

In recent years, bioarrays 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.
However, it is the influence of nonspecific adsorption that becomes a problem when observing the interaction. Non-specific adsorption refers to a case where the target substance adsorbs non-specifically to molecules that do not interact with each other. As a result, a false positive determination is given, which is not preferable. In order to suppress non-specific adsorption, bioarrays in which hydrophilic polymers such as dextran and polyethylene glycol (PEG) are immobilized on the surface have been developed. These hydrophilic polymers are known to have an effect of suppressing nonspecific adsorption of biomolecules.
A biosensor in which non-specific adsorption is suppressed by forming a hydrophilic polymer gel matrix on the biosensor surface is shown. In this method, functional groups are introduced into the gel matrix, and biomolecules are immobilized by covalent bonds using the functional groups. Specifically, the carboxyl group of carboxymethyl dextran was activated with water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the formed succinimide group and the biomolecule were successfully immobilized (Non-patent Document). 1).
However, when a biomolecule is immobilized through a functional group of the gel matrix, it is difficult to measure the phosphorylation reaction of the biomolecule with high sensitivity.
Anal. Biochem. 198, 268, 1991

  The subject of this invention is providing the manufacturing method of the bioarray for suppressing the nonspecific adsorption | suction of a biomolecule, and also increasing the phosphorylation signal with respect to a target peptide.

  As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means, and have reached the present invention.

    1. A method for producing a peptide array for detecting a phosphorylation reaction, wherein the molecular weight of a linking group between the substrate and the amino acid to be phosphorylated of the peptide is 2,500 or less .

    2. Item 2. The method according to Item 1, wherein the linking group has a molecular weight of 1,500 or less.

    3. Item 3. The method according to Item 1 or 2, wherein the linking group has a molecular weight of 1,000 or less.

    4). Item 4. The method according to any one of Items 1 to 3, wherein the linking group has a molecular weight of 500 or less.

    5. Item 5. The method according to any one of Items 1 to 4, wherein the peptide is immobilized via a thiol group.

    6). Item 6. The method according to any one of Items 1 to 5, wherein a heterobifunctional crosslinking agent having a succinimide group or a sulfonic acid succinimide group on one side and a maleimide group on the other side is used.

    7). Item 7. The method according to Item 6, wherein the compound represented by formula (I) is used as a crosslinking agent.

（式中、Ｒは水素原子またはＳＯ₃ ^-(1/nＭⁿ⁺）（Ｍⁿ⁺はｎ価のカチオンを示す。ｎは１又は２を示す。）である)
８． アレイ表面が金であり、金と連結基が結合していることを特徴とする項１〜７のいずれかに記載の方法。

(In the formula, R is a hydrogen atom or SO ₃ ⁻ (1 / nM ^{n +} ) (M ^{n +} represents an n-valent cation; n represents 1 or 2).
8). Item 8. The method according to any one of Items 1 to 7, wherein the array surface is gold, and the gold and the linking group are bonded to each other.

    9. Item 9. The method according to any one of Items 1 to 8, wherein the substrate of the array is a transparent substrate.

    10. Item 10. The method according to any one of Items 1 to 9, wherein two or more kinds of peptides are immobilized on the same array.

    11. Item 11. The method according to any one of Items 1 to 10, wherein at least one terminal of the peptide to be immobilized is a cysteine residue.

    12 Item 12. The method according to any one of Items 1 to 11, wherein the array is used for surface plasmon resonance (SPR) analysis.

  By using the low molecular weight compound in the present invention as a crosslinking agent, a phosphorylation signal of an immobilized peptide is improved, and a measurement system using a highly accurate peptide array in which nonspecific adsorption is also suppressed is obtained. Can do.

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

Phosphorylation of peptides can be performed by applying a test sample that may have protein kinases and a nucleoside triphosphate such as ATP onto the array of the present invention. Optimum phosphorylation reaction conditions vary depending on the type of protein kinase. For example, a test sample that can have protein kinase in a buffer and a nucleoside triphosphate are added, preferably at a temperature of about 10 to 40 ° C. Can be phosphorylated by reacting at a temperature of 30 to 40 ° C. for about 10 minutes to 6 hours, preferably about 30 minutes to 1 hour. If necessary, the phosphorylation reaction solution may contain a substance that assists phosphorylation, such as cAMP, cGMP, Mg ²⁺ , Ca ²⁺ , and phospholipid. In the detection of phosphorylation, the uptake of phosphorus may be confirmed directly, or it is detected by the action of an antibody that recognizes phosphorylated amino acids or a substance that has a specific binding property to a phosphate group. May be.

  Examples of protein kinases targeted for kinetic profiling include enzymes that phosphorylate side chains of amino acids such as tyrosine, serine, threonine, and histidine of proteins. For example, cGMP-dependent protein kinase family, cAMP-dependent protein kinase ( PKA) family, myosin light chain kinase family, protein kinase C (PKC) family, protein kinase D (PKD) family, protein kinase B (PKB) family, protein kinase family belonging to MAP kinase (MAPK) cascade, Src tyrosine kinase family And the receptor tyrosine kinase family.

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

    SFG is a kind of second-order nonlinear optical effect, and is a phenomenon in which two types of incident light (frequency ω1 and frequency ω2) having different frequencies are mixed in a medium to generate light of ω1 + ω2 or ω1−ω2. In particular, when visible light is used as ω1 and wavelength-variable infrared light is used as ω2, vibrational spectroscopy similar to infrared spectroscopy can be performed. Since this method has good surface selectivity, vibrational spectroscopy of molecules at the monomolecular film level is possible, and it is useful as a very sensitive surface analysis method.

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

  The SPR imaging method using this SPR is a method for monitoring the state of interaction between substances by irradiating a polarized light beam over a wide range and analyzing the reflected image by making full use of image processing technology or the like. It is possible to screen an array 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 array with a polarized light beam over a wide range and sufficiently ensuring the illuminance of the light beam is required. The brighter the illuminance of the polarized light flux, the more preferable is the sensitivity of the sensor.

  The type of the light source is not particularly limited, but it is preferable to use light including near infrared light that makes the change in the SPR resonance angle particularly sensitive. Specifically, a white light source capable of irradiating light over a wide range such as a metal halide lamp, a mercury lamp, a xenon lamp, a halogen lamp, a fluorescent lamp, and an incandescent lamp can be used. A halogen lamp that is sufficiently high, has a simple light power supply and is inexpensive is particularly preferable.

  A normal white light source has a defect that light and dark unevenness occurs in the filament portion. If the light from the light source is irradiated as it is, 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 light to the array, 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 an array in which a plurality of substances are immobilized, and to observe the morphology of an object adsorbed on the surface with high sensitivity.

  The array for SPR used in the present invention preferably comprises a metal substrate having a metal thin film formed on a transparent substrate, directly or indirectly on the metal thin film, chemically or physically, a substance or a substance. A slide on which the assembly is fixed is used. The material of the substrate is not particularly limited, but it is preferable to use a transparent material. Specific examples include glass or plastics such as polyethylene terephthalate (PET), polycarbonate, and acrylic. Of these, glass is particularly preferable.

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

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

  One particularly preferred embodiment of the present invention uses an array in which at least one peptide, preferably a plurality of types of peptides, is immobilized on a metal-deposited substrate, and the array It is characterized in that a solution containing a kinase such as a cell lysate is allowed to act, and further, a substance for detecting phosphorylation is allowed to act to detect the state of the interaction, particularly by SPR or SPR imaging. In the present invention, the peptide serving as a substrate for protein kinase refers to a peptide having a property of undergoing phosphorylation by the protein kinase. Here, the substance for detecting phosphorylation is not particularly limited, and examples thereof include antibodies and chelate compounds that recognize phosphorylated amino acids. As the chelate compound, various compounds coordinated with metals such as zinc, gallium, and iron can be used.

  The length of the peptide is not particularly limited, but generally a peptide having 100 amino acid residues or less is used. Preferably, those consisting of 5 to 60 amino acid residues, more preferably about 10 to 25 amino acid residues are used. The peptide may be a peptide obtained by chemical synthesis based on a known technique, or may be a peptide produced by a genetic engineering technique. In addition, in order to facilitate the desorption to the substrate, biotin, a cysteine residue having a thiol group added to one end of the peptide, oligohistidine (His-tag), glutathione-S-transferase It is also useful to use a tag to which a commonly used tag such as (GST) is added.

  The method for immobilizing the peptide on the metal thin film is not particularly limited, but it is more preferable to immobilize the substrate peptide by introducing a functional group that can be easily immobilized on the surface of the metal thin film in advance. preferable. Examples of the functional group include an amino group, a mercapto group, a carboxyl group, and an aldehyde group, and it is particularly preferable to use a mercapto group. In order to introduce these functional groups onto the surface of the metal thin film, it is preferable to use a generally used derivative of alkanethiol.

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

  Regarding the peptide to be immobilized, there may be only one type, or two or more types. Here, the peptide refers to a generally used meaning, and two or more amino acids are linked by peptide bonds. Depending on the purpose of analysis, amino acids in which one or several residues among the amino acid residues are chemically modified may be included. Further, although not particularly limited, it is preferable that at least one peptide having a function as a substrate for a specific enzyme is included.

  Here, the peptide immobilized on the array has a cysteine residue and is linked to the substrate via the SH group of the cysteine residue. 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. Even when it is further added to the sequence, it may be anything that does not prevent phosphorylation. 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 terminal, more preferably only one terminal (N terminal or C terminal). When a cysteine residue is added to one end, only a cysteine residue may be added.

  In the present invention, the molecular weight of the linking group that connects the peptide and the substrate is 2,500 or less. The molecular weight of the linking group is preferably 1,500 or less, more preferably 1000 or less. The structure of the linking group is not particularly limited, but in the case of immobilization via a thiol group, a heterobifunctional crosslinking agent having a succinimide (NHS) group or a sulfonic acid succinimide group and a maleimide (MAL) group Is preferably used.

Specific examples of the heterobifunctional crosslinking agent having an NHS group or a sulfonic acid succinimide group and a MAL group include compounds represented by the formula (I), but are not particularly limited. A compound succinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (hereinafter referred to as SMCC) represented by the formula (I) wherein R is H or R is SO ₃ ⁻ (1 / nM ^{n +} ) (M, n Is as defined above) and is characterized by using a compound Succinosuccinimidyl 4- [N-maleimidemethyl] cyclohexane-1-carboxylate (hereinafter referred to as SSMCC) as a crosslinking agent. In addition, it includes not only compounds having the same structure as the compound represented by formula (I) or formula (II) but also analogized compounds as long as their functions are not impaired. 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.

（式中、Ｒは水素原子またはＳＯ₃ ^-(1/nＭⁿ⁺)（Ｍⁿ⁺はｎ価のカチオンを示す。ｎは１又は２を示す。）である）
1/nＭは、Ｈ，Ｎａ，Ｋ，Ｌｉ，Ｃｓ等のアルカリ金属(ｎ＝１)、ＮＨ₄，Ｎ(Ｍｅ)₄等のＣ₁〜Ｃ₄のアルキル基で置換されていてもよい四級アンモニウム（ｎ＝１）、1/2Ｃａ，1/2Ｍｇ，1/2Ｂａ等のアルカリ金属（ｎ＝２）等が挙げられ、好ましくはアルカリ金属、より好ましくはＮａである。

(In the formula, R is a hydrogen atom or SO ₃ ⁻ (1 / nM ^{n +} ) (M ^{n +} represents an n-valent cation; n represents 1 or 2).
1 / nM may be substituted with an alkali metal (n = 1) such as H, Na, K, Li, or Cs, or a C _{1 to} C ₄ alkyl group such as NH ₄ or N (Me) _4. Alkali metals (n = 2) such as quaternary ammonium (n = 1), 1 / 2Ca, 1 / 2Mg, 1 / 2Ba and the like can be mentioned, and alkali metals are preferable, and Na is more preferable.

  By applying the cross-linking agent of the formula (I) as described above, the phosphorylation signal becomes clearer because the immobilization efficiency of the peptide on the array is significantly higher than that of the high-molecular-weight cross-linking agent. There is an effect. Moreover, there is almost no problem with non-specific effects, and the so-called S / N ratio can be increased.

  In order to form a maleimide surface by introducing SMCC or SSMCC as described above onto the array, the reactivity with the succinimide group at the other end of SMCC or the sulfonic acid succinimide group at the other end of SSMCC It is necessary to introduce a functional group, specifically an amino group, onto the array in advance. The means for introducing an amino group onto the array is not particularly limited. Examples 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 the array. 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 label-free optical detection method, any substance adsorbed on the array is detected as a signal. That is, it is difficult to distinguish nonspecific adsorption of a substance that is not a measurement target from specific adsorption. Therefore, since a means for suppressing non-specific adsorption more severely is required, the method of the present invention 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 desorb from the array 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.

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

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]
(Peptide immobilization)
4 armPEG (SUNBRIGHT PTE-100SH manufactured by NOF Corporation) whose terminal functional group is a thiol group was dissolved in a mixed solution of 7 ml of ethanol: water = 6: 1 at a concentration of 1 mM. The molecular weight of 4armPEG is 10,000, which is a molecule in which four PEG chains having almost the same length from the center are present, and is very hydrophilic. In addition, all four ends of PEG are thiol groups, and particularly exhibit metal binding properties to gold.

  A gold-deposited slide in which 3 nm of chromium was vapor-deposited on an SF15 glass slide of 18 mm square and 2 mm thickness and gold was vapor-deposited at 45 nm was immersed in the 4 armPEG thiol solution for 3 hours to bond 4 armPEG thiol to the entire gold substrate.

  A photomask was placed on the slide and irradiated with a 500 W ultra-high pressure mercury lamp (manufactured by USHIO) for 2 hours to remove 4 armPEG thiol in the UV irradiation part. The photomask has 96 square holes of 500 μm square (consisting of 8 × 12 patterns), and the pitch between the hole centers is designed to be 1 mm. The portion of the photomask with a hole is transparent to UV light and is irradiated onto the slide for patterning. 4armPEG remains in the unirradiated portion, and functions as a reference portion as a background portion of the array.

  It was immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 1 hour to form a self-organized surface of 8-AOT on the UV irradiation part. SSMCC (Pierce) was dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 0.4 mg / ml and reacted with 8-AOT on the gold surface for 15 minutes. The amino group of 8-AOT and the NHS group of SSMCC reacted to introduce an unreacted MAL group on the surface.

On the surface obtained as described above, as shown in the lower part of FIG. 1, a peptide (PKA) consisting of an amino acid sequence serving as a substrate for protein kinase A, a negative control (nPKA; serine residue of protein kinase A substrate) Alanine residue), protein kinase A substrate positive control (pPKA; serine residue phosphorylated), cSrc kinase substrate peptide (cSrc) consisting of phosphate buffer (20 mM phosphate) Acid, 150 mM NaCl; pH 7.2) was dissolved at 1 mg / ml, and spotted 10 nl at a time using a MultiSPRinter spotter (manufactured by Toyobo). Then, the immobilization reaction was carried out by allowing to stand at room temperature for 16 hours in a wet environment. The maleimide group formed on the surface of the array 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) NOF SUNBRIGHT MEH- 50H) is dissolved in a phosphate buffer solution (20 mM phosphate, 150 mM NaCl; pH 7.2) to a concentration of 1 mM, 250 μl is poured onto the array, and allowed to react at room temperature for 1 hour. It was. The molecular weight of PEG thiol used here is 5,000.
(Detection of PKA phosphorylation by SPR analysis)
Phosphorylation by PKA was performed using the array that had been blocked as described above. 400 μl of PKA solution was dropped on the array and reacted at 30 ° C. for 4 hours. The composition of the PKA solution was 1 μl of PKA catalyst subunit (manufactured by Promega), 375 μl of 50 mM Tris-HCl buffer (pH 7.4), 20 μl of 1M magnesium chloride solution, and 4 μl of 10 mM ATP. 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: manufactured by Toyobo), and 50 mM Tris-HCl buffer (pH 7.4) was used as a running buffer. Was flowed into the flow cell at a rate of 100 μl / min. After confirming that the signal from SPR was stabilized, phosphorylated serine antibody PSR-45 (manufactured by Sigma) was injected into the cell in the SPR device to act. The antibodies were allowed to act in order from the one with the highest dilution ratio using solutions diluted 4000 times, 2000 times and 1000 times with the above running buffer. When the signal rise reached a plateau state, the running buffer was fed again for washing. The change of the SPR signal at that time was observed. In addition to the spot site of each substrate, the signal change was also observed in the background.
(Observation results and discussion)
The result of the sensorgram which graphed the SPR signal change is shown in FIG. The sensorgram plots the average value of the signal for each spot. For Background, the average value of signals obtained by using arbitrarily selected locations other than the spot portion of the substrate peptide as a measurement point is plotted. A very strong increase in antibody binding signal was confirmed with respect to the positive control, and a certain level of increase in binding signal was also observed in the PKA substrate. On the other hand, almost no signal change is observed for the negative control, cSrc substrate, blank, and background. Therefore, by this method, phosphorylation of the PKA substrate can be detected very specifically.
[Comparative Example 1]
After introducing 8-AOT on the gold surface, instead of SSMCC, a heterobifunctional polyethylene glycol having a succinimide (NHS) group and a maleimide (MAL) group at the end with a molecular weight of 3400 (NHS-PEG-MAL, manufactured by Nektar) Except for the point of acting), all were examined in the same manner as in Example 1. NHS-PEG-MAL was dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 10 mg / ml and reacted with 8-AOT on the gold surface for 2 hours. The results are shown in FIG.

  In this case, the binding signal itself is weak as a whole. Furthermore, non-specific signals have been confirmed in addition to PKA substrates, which is an undesirable result in terms of specificity.

  By the method of the present invention, it is possible to amplify the phosphorylation signal in the peptide array, while suppressing non-specific adsorption, and more accurate measurement is possible. The processing method is also very easy, and is expected to contribute greatly to the industry.

It is a figure which shows the result of the SPR analysis in Example 1. It is a figure which shows the result of the SPR analysis in the comparative example 1.

Claims (6)

Surface Plasmon Resonance (SPR) for the detection of phosphorylation of a peptide fixed on the array by detecting antibodies binding signal by the analysis, a process for the preparation of peptide arrays array surface is gold,
(A) using a peptide having at least one terminal cysteine residue and 5 to 25 amino acid residues ;
(B) the molecular weight of the linking group between the substrate and the amino acid to be phosphorylated of the peptide is 500 or less ; and
(C) By using a heterobifunctional crosslinking agent having a succinimide group or a sulfonic acid succinimide group on one side and a maleimide group on the other side, the peptide is arrayed via the thiol group of the cysteine residue of (A). A method for producing a peptide array, characterized in that the peptide array is immobilized . The method according to claim 1 , wherein the compound represented by the formula (I) is used as a crosslinking agent.

(In the formula, R is a hydrogen atom or SO ₃ ⁻ (1 / nM ^{n +} ) (M ^{n +} represents an n-valent cation; n represents 1 or 2). The method according to claim 1 or 2 , wherein the substrate of the array is a transparent substrate. The method according to any one of claims 1 to 3, two or more peptides, characterized in that it is immobilized on the same array. Surface Plasmon Resonance (SPR) for the detection of phosphorylation of immobilized peptide array by detecting antibodies binding signal by analyzing a gold array surface, and antibody binding by surface plasmon resonance (SPR) analysis A peptide array for detecting a phosphorylation reaction of a peptide by detecting a signal ,
(A) At least one terminal of the peptide is a cysteine residue and the number of amino acid residues is 5 to 25 ,
(B) the molecular weight of the linking group between the substrate and the amino acid to be phosphorylated of the peptide is 500 or less ; and
(C) By using a heterobifunctional crosslinking agent having a succinimide group or a sulfonic acid succinimide group on one side and a maleimide group on the other side, the peptide is arrayed via the thiol group of the cysteine residue of (A). A peptide array characterized by being immobilized on . The peptide array according to claim 5 , which is used for detecting a phosphorylation reaction of a peptide by detecting an antibody binding signal by surface plasmon resonance (SPR) analysis.

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