JP2006141338A - Method for detecting phosphorylation on basal plate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a peptide chip capable of obtaining data with high reliability, by controlling nonspecific adsorption of a biomolecule, while amplifying a phosphorylation signal, in a system for measuring phosphorylation by a radioisotope for which the peptide chip is used. <P>SOLUTION: An array for detecting the phosphorylation uses the radioisotope, wherein a protein or a peptide is fastened to a basal plate of the array by using a low-molecular compound having a molecular weight of ≤2,000, for example, a compound expressed by formula (I) or formula (II), as a crosslinking agent. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an array in which proteins or peptides used for phosphorylation detection detection are immobilized using a radioisotope (hereinafter also referred to as RI). More particularly, the present invention relates to an array that can increase phosphorylation signal and reduce non-specific effects by using a low molecular weight compound as a cross-linking agent.

  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. In recent years, various techniques have been reported as techniques for comprehensively analyzing phosphorylation. In particular, phosphorylation assays using RI can directly monitor phosphorus uptake, and are also advantageous in terms of sensitivity and specificity, and various reports have been made (Non-Patent Documents 1 to 4, etc.). .

  The problem in observing the interaction between these biomolecules is sensitivity and the influence of nonspecific adsorption, that is, specificity. Here, nonspecific adsorption refers to the case where the target substance adsorbs nonspecifically to molecules that would otherwise not interact. As a result, a false positive determination is given, which is not preferable. In order to suppress nonspecific adsorption, a biochip has been developed in which a substrate on which a biopolymer is immobilized is coated with a hydrophilic polymer such as dextran or polyethylene glycol (PEG). For example, the carboxyl group of carboxymethyldextran is activated with water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the formed succinimide group and the biomolecule are successfully immobilized (Non-patent Document 5). . These hydrophilic polymers are known to have an effect of suppressing nonspecific adsorption of biomolecules. However, the coating of the hydrophilic polymer may cause a problem that the biomolecule is buried in the hydrophilic polymer and the interaction between the biopolymers is inhibited.

This inhibition of the interaction between biopolymers causes a decrease in the measurement signal. This inhibition of interaction is not only due to the coating of the hydrophilic polymer, but is often caused by binding the biopolymer directly to the substrate. This is because when the biopolymer is directly bonded to the substrate, the degree of freedom of the biopolymer is suppressed. In order to improve the measurement signal of the biochip, it is necessary to facilitate the interaction between biopolymers.

In order to facilitate interaction between biomolecules, studies have been made to immobilize biomolecules on a substrate via a spacer. However, immobilization of a biomolecule through such a hydrophilic polymer is not always preferred because it may cause adverse effects such as a decrease in immobilization reaction efficiency and inhibition on the measurement system. In particular, when immobilizing a peptide on a substrate, the yield of the immobilization reaction via a high molecular weight linker is greatly reduced, resulting in a decrease in measurement signal and an increase in nonspecific signal. Seen frequently.

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 Anal. Biochem. 198, 268, 1991

  The present invention not only can remarkably increase the efficiency of immobilization on a chip without denaturing proteins and peptides, but also can effectively suppress non-specific adsorption and increase the phosphorylation signal. To provide an array.

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. An array for simultaneously detecting phosphorylation of a plurality of types of proteins or peptides using a radioisotope, wherein the proteins or peptides are immobilized on a substrate via a crosslinking agent having a molecular weight of 2,000 or less An array characterized by that.
2. 1. The array according to 1, which is immobilized through a crosslinking agent having a molecular weight of 1,000 or less.
3. The array of 1 or 2, which is immobilized through a cross-linking agent having a molecular weight of 500 or less.
4). 4. The array according to any one of 1 to 3, wherein the crosslinking agent is a heterobifunctional crosslinking agent having a succinimide group or a sulfated succinimide group on one side and a maleimide group on the other side. 5. The array according to any one of 1 to 4, wherein a compound represented by formula (I) or formula (II) is used as a crosslinking agent.
6). The array according to any one of 1 to 5, wherein a peptide having a cysteine residue at one end is immobilized.
7). The array according to any one of 1 to 6, wherein the surface of the substrate is gold.
8). The array according to any one of 1 to 7, wherein the substrate is glass.
Using the array of any one of 9.1 to 8, phosphorylation on a substrate characterized in that ³² P or ³³ P is allowed to act as a nuclide on a test material that may contain a protein kinase. Detection method.
10. 9. A method for detecting phosphorylation on a substrate according to 9, wherein a blocking treatment is performed before the sample material containing protein kinase can be allowed to act.
11. A method for detecting phosphorylation on 10 substrates, wherein a blocking treatment is performed using a hydrophilic polymer.

  Measurement using an accurate peptide chip that improves the phosphorylation signal of immobilized protein or peptide by RI and also suppresses nonspecific adsorption by using the low molecular weight compound in the present invention as a crosslinking agent. A system can be obtained.

The array according to the present invention preferably comprises a metal substrate having a metal thin film formed on a transparent substrate, and a substance or a collection of substances is fixed directly or indirectly, chemically or physically on the metal thin film. The slides are 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. Although the thickness of a board | substrate is not specifically limited, About 0.1-20 mm is preferable and about 1-2 mm is more preferable.

  It is possible to immobilize the protein or peptide as it is on the substrate, but there is also a method in which a metal thin film is coated on the surface and immobilized thereon. 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 not particularly limited, but is preferably about 10 to 3000 mm, more preferably about 100 to 600 mm.

Phosphorylation of the immobilized protein or peptide can be performed by applying a test sample capable of having protein kinase and RI-labeled nucleoside triphosphate such as ATP on the array of the present invention. As the various types of RI to be labeled, ³² P or ³³ P is usually used, but ³³ P is more preferable in terms of both sensitivity and specificity. Optimum phosphorylation reaction conditions vary depending on the type of protein kinase. For example, a test sample that can have protein kinase in a buffer and a nucleoside triphosphate are added, preferably at a temperature of about 10 to 40 ° C. Can be phosphorylated by reacting at a temperature of 30 to 40 ° C. for about 10 minutes to 6 hours, preferably about 30 to 1 hour. If necessary, the phosphorylation reaction solution may contain a substance that assists phosphorylation, such as cAMP, cGMP, Mg ²⁺ , and Ca ²⁺ phospholipid. In the detection of phosphorylation, autoradiography can be performed for direct phosphorus uptake and the detection can be performed using an image analyzer.

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, the purpose of the protein or peptide to be immobilized is to comprehensively profile the dynamics of protein kinases, and therefore one kind of peptide is phosphorylated only by one kind of protein kinase (ie, a specific protein or peptide). It functions as a substrate for protein kinases.) More preferably, it is not phosphorylated by other protein kinases. Either a protein or a peptide may be used, but a peptide is more advantageous than a protein in that it can function biologically without depending on secondary structure. In recent years, techniques for synthesizing many types of peptides at high speed have also been advanced, which is advantageous in preparing a large number of substrates for comprehensive analysis. The amino acid sequence serving as the substrate for the protein kinase is known or can be appropriately selected based on the known sequence. If the array of the present invention has immobilized proteins or peptides of a type corresponding to a plurality of types of protein kinases that need to know the dynamics, all protein kinases can be profiled with a single array. it can.

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

The method for immobilizing the peptide on the metal thin film is not particularly limited, but it is more preferable to immobilize the substrate peptide by introducing a functional group that can be easily immobilized on the surface of the metal thin film in advance. preferable. Examples of the functional group include an amino group, a mercapto group, a carboxyl group, and an aldehyde group, 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 through an alkanethiol layer and modifying the background with PEG (polyethylene glycol) may be used. Good. In addition, in order to further suppress non-specific effects, it is also possible to immobilize a peptide after binding a derivative in which a functional group as described above is introduced to the end of PEG to alkanethiol, which also exerts a spacer effect. It is useful in.

With respect to the peptide to be immobilized, depending on the purpose of analysis, amino acids in which one to several residues among the amino acid residues are chemically modified may be included. 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 case where the functional group is present in the peptide specifically refers to a state in which at least one cysteine residue is present in the amino acid sequence of the peptide to be immobilized. The cysteine residue is an amino acid necessary for the peptide to be immobilized to have its original function, even if it exists as an essential residue as an amino acid sequence necessary for the peptide to function. It may be a case where it is further added to the sequence. The position of the cysteine residue in the peptide to be immobilized is not particularly limited, but it is preferably added to at least one end, more preferably only to one end. When a cysteine residue is added to one end, only a cysteine residue may be added, but a spacer is used to increase the interaction efficiency with the substance to be acted on 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.

Moreover, the case where the functional group is added to the peptide means that the compound having a thiol group is chemically bonded to one or more amino acid residues to the peptide to be immobilized. Say. The bonding position of the compound is not particularly limited, but is preferably bonded to any terminal amino acid residue.

  In the present invention, a compound having a molecular weight of 2,000 or less is used for immobilizing a peptide. The molecular weight is preferably 1,000 or less, more preferably 500 or less. The structure of the compound is not particularly limited, but when immobilizing via a thiol group, a heterobifunctional crosslinking agent having a succinimide (NHS) group or a succinimide sulfate group and a maleimide (MAL) group is used. Is preferred.

  Specific examples of the heterobifunctional crosslinking agent having an NHS group or a succinimide sulfate group and a MAL group include compounds represented by the formula (I) or the formula (II), but are not particularly limited. Compound Succinimidyl 4- [N-maleimidomethyl] 4- [N-maleimidomethyl] cyclohexene-1-carboxylate (hereinafter referred to as SMCC) or compound Sulfocinimidyl 4- [N-maleimidomethyl] Hereinafter, it is referred to as SSMCC.) As a cross-linking agent. In addition, it includes not only a compound having the same structure as the compound represented by formula (I) or formula (II) but also an analogized compound as long as its function is 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.

  By applying a low molecular weight compound as described above, the phosphorylation signal becomes clearer because the efficiency of immobilization of the peptide on the chip is significantly higher than when a high molecular weight substance is used as a crosslinking agent. This is an effect. Further, 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 chip, it has reactivity with the succinimide group at the other end of SMCC or the succinimide sulfate group at the other end of SSMCC. It is necessary to introduce a functional group, specifically 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 interaction analysis method as described above, it is important to distinguish between non-specific adsorption of a substance that is not a measurement target and specific adsorption. Since a means for suppressing non-specific adsorption more severely is required, the immobilization method of the present invention is very effective.

  The blocking treatment for suppressing nonspecific adsorption is preferably performed using a hydrophilic polymer. In the ELISA method or the like, 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. It is preferable to perform blocking by covalent bond. 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.

One particularly preferred embodiment of the present invention uses an array in which a plurality of peptides serving as protein kinase substrates are immobilized on a metal-deposited substrate, and γ ³³ P such as a cell disruption solution is used in the array. -ATP together by the action of solutions containing protein kinases, thoroughly washed array, after drying, characterized in that directly detects the ^{33 P} incorporation by autoradiography. 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.

  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 approximately the same length from the center are present, and is very hydrophilic. In addition, all four ends of PEG are thiol groups, and particularly exhibit metal binding properties to gold.

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

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

  It was immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 1 hour to form a self-organized surface of 8-AOT on the UV irradiation part. SSMCC (Pierce) was dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 0.4 mg / mL and reacted with 8-AOT on the gold surface 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, 72 kinds of synthetic peptides as shown in Table 1,
All were dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 1 mg / mL, and spotted 10 nL at a time using a MultiSPRinter spotter (manufactured by Toyobo). FIG. 1 shows a spot pattern. Then, the immobilization reaction was carried out by allowing to stand at room temperature for 16 hours in a wet environment. The maleimide group formed on the surface of the chip reacts with the thiol group possessed by the cysteine residue at the terminal of the substrate peptide, so that the substrate peptide can be covalently immobilized on the surface.

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

(Detection of phosphorylation by autoradiography)
Using the array subjected to blocking as described above, phosphorylation with various protein kinases was performed. 400 μL of protein kinase solution was dropped on the array and reacted at 30 ° C. As a reaction buffer, 50 mM Tris-HCl buffer (50 mM magnesium chloride; pH 7.4) was used. Incidentally, γ ³³ P-ATP in the reaction mixture; the (9.25 MBq Amersham Biosciences) The reaction was carried out 2μL added. Protein kinases include PKA catalytic subunit (Promega), PKCβI (Sigma), PKCδ (Sigma), PKCζ (Sigma), MAPK (ERK2; Stratagene), p38 (Biomol), Akt (Up) In each case, the stock solution was diluted 400 times with the above reaction buffer. The reaction time was 1 hour for PKA, PKCβI, PKCδ, PKCζ and Akt, and 1.5 hours for ERK2 and p38. In the PKCβI reaction, 0.1 mM calcium chloride, 8 μg / mL phosphatidylserine (manufactured by Sigma) and 0.8 μg / mL diacylglycerol (manufactured by Sigma) were further added to the reaction solution described above. . In the PKCδ reaction, phosphatidylserine having a concentration of 8 μg / mL and diacylglycerol having a concentration of 0.8 μg / mL were added. In addition, regarding PKA and PKCζ reaction, a reaction by mixed kinase (30 ° C., 1 hour) with different mixing ratios was also examined.

  The array that had been phosphorylated was washed with PBS and water three times, and the surface of the array was sufficiently dried, followed by exposure to an IP sheet (manufactured by Fuji Photo Film). The exposure time was 15 minutes to 2 hours. Thereafter, reading was performed using an image analyzer BAS1800II.

(Observation results and discussion)
FIG. 2 shows a comparison result of phosphorylation patterns by the PKA and PKCζ reactions. Both have distinctly different phosphorylation patterns. FIG. 3 shows a comparison result of phosphorylation patterns of mixed kinases of PKA and PKCζ with three mixing ratios. It was recognized that the phosphorylation pattern changed greatly when the mixing ratio was changed. This result suggests the possibility of estimating expression patterns of various protein kinases by profiling using the array of the present invention. FIG. 4 shows the comparison results of phosphorylation patterns by the PKC reaction of three types of subtypes. It is confirmed that the phosphorylation pattern varies greatly depending on the subtype. This suggests the possibility of analyzing the expression pattern of the kinase subtype by performing profiling using the array of the present invention.

FIG. 5 shows comparison results of phosphorylation patterns by two types of MAPK reactions. Both MAPK reactions show very strong phosphorylation in two types of peptides. FIG. 6 shows the phosphorylation pattern by the Akt kinase reaction. A fairly strong phosphorylation was observed in several types of peptides. These results are largely different from the PKA and PKC reactions as phosphorylation patterns. As described above, it was confirmed that phosphorylation patterns differed greatly by the action of various protein kinases. From this result, it was confirmed that the array of the present invention was very useful in conducting phosphorylation assay using RI.

  According to the present invention, while it is possible to amplify the phosphorylation signal due to RI on the array, nonspecific adsorption can be sufficiently suppressed, and more accurate measurement is possible. In addition, since the processing method is very easy, it is expected to greatly contribute to the industry.

It is a figure which shows the spot pattern of the peptide array used in the Example. In an Example, it is a figure which shows the phosphorylation pattern by PKA and PKCζ reaction. In an Example, it is a figure which shows the phosphorylation pattern by mixed kinase reaction of PKA and PKCζ which changed the mixing ratio. In an Example, it is a figure which shows the phosphorylation pattern by PKC reaction from which a subtype differs. In an Example, it is a figure which shows the phosphorylation pattern by MAPK reaction. In an Example, it is a figure which shows the phosphorylation pattern by Akt kinase reaction.

Claims (11)

An array for simultaneously detecting phosphorylation of a plurality of types of proteins or peptides using a radioisotope, wherein the proteins or peptides are immobilized on a substrate via a crosslinking agent having a molecular weight of 2,000 or less An array characterized by that. The array according to claim 1, wherein the array is immobilized via a cross-linking agent having a molecular weight of 1,000 or less. The array according to claim 1 or 2, which is immobilized through a cross-linking agent having a molecular weight of 500 or less. The array according to any one of claims 1 to 3, wherein the crosslinking agent is a heterobifunctional crosslinking agent having a succinimide group or a sulfated succinimide group on one side and a maleimide group on the other side. The array according to any one of claims 1 to 4, wherein a compound represented by formula (I) or formula (II) is used as a crosslinking agent.
The array according to any one of claims 1 to 5, wherein a peptide having a cysteine residue at one end is immobilized. The array according to claim 1, wherein the surface of the substrate is gold. The array according to claim 1, wherein the substrate is glass. Using the array according to any one of claims 1 to 8, phosphorous on a substrate, wherein ³² P or ³³ P is allowed to act as a nuclide on a test material that may contain protein kinase. Detection method of oxidation. 10. The method for detecting phosphorylation on a substrate according to claim 9, wherein a blocking treatment is performed before the sample material containing protein kinase can be allowed to act. The method for detecting phosphorylation on a substrate according to claim 10, wherein a blocking treatment is performed using a hydrophilic polymer.