JP2007143421A - Method for detecting on-chip phosphorylation using radioactive substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a system for on-chip evaluation of a protein kinase activity responsive even to a high through-put for drug development screening by a simple method and having especially excellent aspects of specificity. <P>SOLUTION: A method for detecting phosphorylation is carried out as follows. A solution containing the protein kinase or considered to contain the protein kinase is made to act on a chip having a gold surface in which a peptide or a protein is immobilized to thereby detect the phosphorylation of the peptide or protein by the method for using an R (radioactive substance). In the process, the phosphorylation reaction on the chip with the solution is carried out and an enzyme treatment is then conducted. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for detecting on-chip phosphorylation using a radioactive substance (hereinafter also referred to as RI). More specifically, when detecting phosphorylation of the peptide or protein by RI on a chip on which the peptide or protein is immobilized, by reacting with a solution containing or thought to contain protein kinase, nonspecificity is detected. The present invention relates to a method for detecting a phosphorylation inhibitory activity for reducing the adverse effect.

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

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

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

  An object of the present invention is to provide an evaluation system for detecting protein kinase activity in On-chip with high specificity, which is capable of high throughput for drug discovery screening by a simple 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) A method of using a radioactive substance to phosphorylate a peptide or protein by allowing a solution containing or considered to contain a protein kinase to act on a chip having a gold surface on which the peptide or protein is immobilized A phosphorylation detection method comprising: performing a phosphorylation reaction on a chip with the solution and then performing an enzyme treatment.
(2) The method for detecting phosphorylation according to (1), wherein the enzyme is a protease.
(3) The method for detecting phosphorylation according to (1) or (2), wherein the enzyme is trypsin.
(4) The phosphorylation detection method according to any one of (1) to (3), wherein the chip surface is gold.
(5) The method for detecting phosphorylation according to any one of (1) to (4), wherein the nuclide of the radioactive substance is ³² P or ³³ P.
(6) The phosphorylation detection method according to any one of (1) to (5), wherein the method using a radioactive substance is autoradiography.
(7) The method for detecting phosphorylation according to any one of (1) to (6), wherein the peptide or protein is immobilized on the chip via a thiol group.
(8) The method for detecting phosphorylation according to any one of (1) to (7), wherein a peptide having 30 or less amino acids is immobilized on a chip.

  By the array analysis using RI in the present invention, it is possible to obtain a measurement system for protein kinase inhibition test with On-chip. It is also useful to be able to cope with high throughput by performing analysis with an array. In particular, the pharmaceutical industry is expected to be very useful as an evaluation system for novel drug discovery screening such as kinase inhibitors.

  The present invention is characterized in that protein kinase activity is evaluated on-chip by array analysis using RI. By using RI, a phosphorylation reaction can be directly monitored, and a detection system excellent in both sensitivity and specificity can be obtained. The chip surface may be a substrate such as glass or plastic, or a film such as cellulose. However, the use of a gold surface makes surface modification easier than using a general glass surface, and is nonspecific. There is an advantage that the influence is easy to be reduced.

  The material of the substrate surface used in the present invention is preferably gold that 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. Alternatively, plastics such as polyethylene terephthalate (PET), polycarbonate, and acrylic are also included. Transparent glass is easily available and is advantageous in terms of versatility. When a glass substrate is used, the type of glass and the thickness of the substrate are not particularly limited, but the thickness is preferably about 0.1 to 20 mm, for example, a substrate of about 1 to 2 mm is used. Although it does not specifically limit regarding a magnitude | size or a shape, You may use things like the slide glass marketed normally.

  As a method for forming a metal substrate, a method of coating a gold thin layer is preferable. The method for coating gold 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.

  In the present invention, an array in which peptides or proteins are immobilized on a substrate as described above is used. As the peptide or protein, at least one containing an amino acid sequence capable of functioning as a protein kinase substrate, preferably a plurality of amino acid sequences that are phosphorylated by different types of protein kinases are used. Furthermore, it is preferable that a 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 30 residues or less, and more preferably about 10 to 25 residues.

  The method for immobilizing peptides or proteins in the array used in the present invention is not particularly limited, and examples include a method using an amino group or thiol group in a peptide sequence, a method using a His tag or a GST tag. Among these, the method of immobilizing via a thiol group is particularly preferable from the viewpoints of specificity and sensitivity.

  It is preferable that at least one cysteine residue is present in the amino acid sequence of the peptide to be immobilized. The cysteine residue is an amino acid necessary for the peptide to be immobilized to have its original function, even if it exists as an essential residue as an amino acid sequence necessary for the peptide to function. It may be a case where it is further added to the sequence. The position of the cysteine residue in the peptide to be immobilized is not particularly limited, but it is preferably added to at least one end, more preferably only to one end. When a cysteine residue is added to one end, only a cysteine residue may be added, but a spacer is used in order to increase the efficiency of interaction with a substance that acts by increasing the degree of freedom of the immobilized peptide. As an amino acid sequence of 1 to several residues may be further added. The amino acid sequence of the spacer portion is not particularly limited, but it is particularly preferable to add a sequence having 1 to several glycine residues and / or alanine residues or serine residues.

  Moreover, you may use the state in which the compound which has a thiol group is chemically combined in the amino acid residue in one or more places with respect to the peptide fix | immobilized. The bonding position of the compound is not particularly limited, but is preferably bonded to any terminal amino acid residue.

  In the present invention, when a peptide is immobilized via a thiol group, a heterobifunctional cross-linking agent having a succinimide (NHS) group or a sulfated succinimide group and a maleimide (MAL) group is introduced after introducing an amino group to the surface in advance. It is particularly preferable to use it. As such a heterobifunctional cross-linking agent, it is possible to use a hydrophilic polymer such as PEG in which both ends are modified with NHS groups and MAL groups, but the immobilization yield of the peptide is improved. In order to achieve this, a lower molecular weight 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.

  By applying low molecular weight compounds as described above, the efficiency of immobilizing peptides on the chip is significantly higher than when high molecular weight substances are used as cross-linking agents. Thus, there is an effect 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 a chip to form a maleimide surface, a succinimide group at the other end of the SMCC or a functional group reactive with the succinimide sulfate group at the other end of the SSMCC, Specifically, it is necessary to introduce an amino group on the chip in advance. The means for introducing an amino group on the chip is not particularly limited. Examples thereof include a self-assembled surface method for aligning molecules on the substrate surface, a method of introducing using a reaction reagent, and a means for coating a substance having a functional group on a chip. Also included are means for introducing an amino group using a cross-linking agent starting from the functional group introduced on the surface.

  In the array used in the present invention, the non-immobilized portion (background portion) of the peptide or protein is preferably coated with a compound represented by the formula (III). In formula (III), 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.

  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.

An object of the present invention is to detect phosphorylation activity using RI. As the nuclide of RI, ³² P or ³³ P can be mentioned, but ³³ P is more preferable from the viewpoint of excellent sensitivity, a slightly longer half-life and stability of the reagent itself. When a solution containing or considered to contain a 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 allowed to coexist in the solution. become. The protein kinase to be subjected to profiling may be a commercially available reagent, but it is also possible to use a protein kinase already contained in or estimated to be contained in a cell-derived extract.

For example, by immobilizing a protein kinase reagent or nucleoside triphosphate (ATP), which is already contained in a buffer or cell extract or a mixture of both, and acting on the array directly, the immobilized substrate Can be phosphorylated. In this case, ATP labeled with RI is used. For example. Examples thereof include γ ³² P-ATP and γ ³³ P-ATP. From the viewpoint of the stability of the reagent, γ ³³ P-ATP is more preferable. 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 peptide or protein can be phosphorylated. If necessary, a substance that assists or promotes phosphorylation such as cAMP, cGMP, Mg ²⁺ , and Ca ²⁺ may coexist in the reaction solution.

  In the present invention, when detecting phosphorylation, the chip surface is treated with an enzyme. Examples of the enzyme include a protease having an action of degrading proteins and peptides. The protease enzyme is not particularly limited, and specific examples include pepsin, trypsin, chymotrypsin, papain, ficin, bromelain and the like. Of these, trypsin is particularly preferable.

  The enzyme treatment is preferably carried out by preparing an enzyme having an appropriate concentration in a buffer solution having an appropriate composition and then dropping it onto a washed and dried chip after the phosphorylation reaction. The treatment temperature is preferably 30 to 40 ° C., more preferably 30 to 37 ° C. The treatment time is preferably 5 to 60 minutes, more preferably 10 to 30 minutes.

  Autoradiography is effective for detecting phosphorylation on the array. After sufficiently washing the array that has undergone phosphorylation, it can be dried and exposed to a dedicated film or sheet and analyzed by a dedicated image reader. This method is a very useful technique in that phosphate uptake can be directly observed for a detection system using an antibody or the like. It is also the most excellent method from the viewpoint of sensitivity and specificity.

  As described above, exhaustive analysis by profiling of protein kinase activity can be performed by conducting a phosphorylation reaction on-chip by the method of the present invention. In particular, by monitoring inhibitory activity against protein kinases, it is possible to provide a useful technique in drug discovery screening.

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

[Example 1]
(Production of array)
As shown in the formula (IV), a linear thiol PEG reagent (SPSPT-0011 manufactured by SensoPath) having a terminal functional group as 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 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, the maleimide group could be introduced to the surface via PEG.

  On the surface obtained as described above, as shown in FIG. 1, a peptide consisting of an amino acid sequence serving as a substrate for PKA (protein kinase A) (SEQ ID NO: 1), a PKA substrate negative control (SEQ ID NO: 2) A serine residue is substituted with an alanine residue), a positive control of a PKA substrate (the serine residue of SEQ ID NO: 1 is phosphorylated), a peptide comprising the amino acid sequence shown in SEQ ID NO: 3 as a PKC substrate, and its negative control ( Both the positive control (SEQ ID NO: 4: serine residue of SEQ ID NO: 3 was substituted with an alanine residue) and the positive control (serine residue of SEQ ID NO: 3 were phosphorylated) were both phosphate buffer (20 mM phosphate). , 150 mM NaCl; pH 7.2) at 1 mg / ml, and MultiSPRinter ( Spotted 10 nl at a time using a registered spotter (manufactured by Toyobo Co., Ltd.), and then allowed to stand for 16 hours at room temperature in a wet environment to carry out an immobilization reaction Maleimide formed on the surface of the chip The group and the thiol group possessed by the cysteine residue at the terminal of the substrate peptide react to immobilize the substrate peptide on the surface covalently.

(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 PKCδ by autoradiography-1)
Phosphorylation by PKCδ was performed on the array that had been blocked as described above. As PKCδ, Protein Kinase Cδ Isozyme (manufactured by Sigma) was used at a concentration of 0.5 μg / ml. The reaction solution composition includes [γ ³³ P] -ATP (50 uCi / ml; manufactured by Amersham Biosciences), 50 mM MOPS buffer solution containing 50 mM MgCl ₂ , 8 μg / ml phosphatidylserine, 0.8 μg / ml diacylglycerol ( pH 7.4) was used. 300 μl of the PKCδ solution was dropped on the substrate-immobilized region of the array to sufficiently spread the solution, and reacted at 30 ° C. for 1 hour.

Thereafter, the array was washed three times with PBS and water, and the array surface was dried. Then, a 0.25% trypsin solution (manufactured by Nacalai Tesque) was treated at 30 ° C. for 10, 20, and 30 minutes. A case where no trypsin treatment was performed was used as a control. After the trypsin treatment for a predetermined time, the array was washed three times with PBS and water, and the array surface was dried. (Fuji Photo Film) was used. The exposure was carried out for 30 minutes using a sheet dedicated to the apparatus (SG imaging plate manufactured by Fuji Photo Film). The results are shown in FIG. As the trypsin treatment time is longer, the background reduction effect is remarkably confirmed. It is also confirmed that nonspecific ³³ P incorporation in the negative control substrate of alanine is also reduced. When PKC is allowed to act, it tends to be adsorbed particularly on the surface of the array, and it is considered that non-specific influences due to self-phosphorylation are reduced.

[Example 3]
(Detection of phosphorylation by PKCδ by autoradiography-2)
In the same manner as in Example 1, a similar array was prepared and subjected to blocking treatment. Thereafter, a phosphorylation reaction with PKCδ was performed with On-Chip under the same conditions as in Example 2. The array was washed three times with PBS and water, the array surface was dried, and then trypsinized. The treatment conditions were 10 minutes at 30 ° C., and the trypsin concentrations were 0.00025% and 0.0025%. A case where no trypsin treatment was performed was used as a control. After trypsin treatment for a predetermined time, the array was washed three times with PBS and water, the array surface was dried, and the incorporation of RI in each immobilized substrate by autoradiography as in Example 2 The result of reading was shown in FIG. Although there is a difference in the suppression effect of non-specific influence depending on the trypsin concentration, it is recognized that the effect is somewhat effective even at a 1 / 100th trypsin concentration in the case of Example 2.

  By utilizing the present invention, a wide variety of protein kinase signals can be analyzed comprehensively and efficiently with excellent specificity, and protein kinases in cells accompanying the introduction of genes with unknown functions or drug administration Dynamic profiling can be done effectively. 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 the arrangement of each immobilized peptide in the peptide array prepared in Example 1. In Example 2, it is a figure which shows the result of having observed the On-chip phosphorylation by PKC (delta) by autoradiography. In Example 3, it is the figure which showed On-chip phosphorylation by PKC (delta) by the change of RI uptake | capture amount by autoradiography.

Claims (8)

  On a chip having a gold surface on which a peptide or protein is immobilized, phosphorylation of the peptide or protein is detected by a method using a radioactive substance by allowing a solution containing or considered to contain a protein kinase to act. In this case, a phosphorylation detection method comprising performing a phosphorylation reaction on a chip with the solution and then performing an enzyme treatment.   The method for detecting phosphorylation according to claim 1, wherein the enzyme is a protease.   The method according to claim 1 or 2, wherein the enzyme is trypsin.     The method for detecting phosphorylation according to claim 1, wherein the chip surface is gold. The method for detecting phosphorylation according to claim 1, wherein the nuclide of the radioactive substance is ³² P or ³³ P.   The method for detecting phosphorylation according to claim 1, wherein the method using a radioactive substance is autoradiography.   The method for detecting phosphorylation according to any one of claims 1 to 6, wherein the peptide or protein is immobilized on the chip via a thiol group.   The method for detecting phosphorylation according to any one of claims 1 to 7, wherein a peptide having 30 or less amino acids is immobilized on a chip.