JP2009052908A - Detection method of caspase activity on substrate by surface plasmon resonance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inclusive analyzing technique related to caspase activity. <P>SOLUTION: An array on which a plurality of peptides are immobilized is used to analyze caspase activity by surface plasmon resonance. Especially biotinized peptide, preferably peptide, in which a hydrophilic linker is inserted, is used and the bonding signal of streptoavidin is reduced to detect caspase activity. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for detecting caspase activity on a chip by using surface plasmon resonance (SPR). More specifically, the present invention relates to a method for detecting caspase activity by allowing a solution (sample) containing or thought to contain caspase to act on a chip on which a peptide is immobilized.

  Caspase is an enzyme that forms the basis of apoptotic signal transduction, and is one of important protease enzymes. Apoptosis is a concept proposed by Kerr et al. And means cell death controlled by a gene in order to maintain the life of an individual. This plays an important role not only in morphogenesis in ontogenesis and establishment of a neural network, but also in maintenance of homeostasis and establishment of immune system by cell replacement and endocrine system in mature individuals. It is also closely related to the onset of cancer, autoimmune diseases, viral infections such as AIDS, and neurodegenerative diseases such as Alzheimer's disease. In the execution process of apoptosis, limited degradation of specific proteins (PARP, ICAD, Acinus, etc.) by caspase plays a central role (Biochem. J. 326, 1-16 (1997)). Many types of caspases have already been reported, and the mechanisms for their activation are being elucidated. Establishing a technique for comprehensively analyzing the activity of these caspases has important implications in fields such as drug development and diagnosis.

  Measurement of protease activity, including caspases, quantifies the degradation of substrate proteins and peptides by ultraviolet absorption, and the amount of labeling substances released using substrates labeled with fluorescent substances, radioactive substances, etc. It is usually done by the method of doing. However, such a method cannot cope with the high throughput demanded in recent years in fields such as proteomics research.

  A method using a fluorescent array has been reported as a caspase assay system that may be compatible with high throughput (Non-Patent Documents 1-3). Although this method enables highly sensitive detection, there are problems with reproducibility and data stability, and there are problems with quantitative assays.

Recently, as disclosed in, for example, Patent Document 1, as a method using surface plasmon resonance (SPR), a method of detecting the mass of an intact substrate versus the mass of a cleaved substrate has been disclosed. However, this method is a method in which an enzyme reaction is carried out in a separate reaction vessel in advance, and then the substrate is immobilized on a chip and used for SPR measurement, although it is not necessarily a method that can sufficiently cope with high throughput. Absent. Further, Patent Document 2 discloses a method in which streptavidin (SA) is bound to a biotinylated substrate peptide and then a caspase is allowed to act to monitor a decrease in SPR signal due to hydrolysis. . This method has a problem in stability and reproducibility of data because access to the immobilized peptide of caspase due to steric influence by binding SA is likely to be hindered.
Japanese Patent No. 2968048 JP 2004-357642 A J. et al. Am. Chem. Soc. 124, 14868, 2002 Bioorg. & Med. Chem. Lett. 9, 1667, 1999 Combinatorial Chemistry & High Throughput Screening 9, 21, 2006

  An object of the present invention is to provide an evaluation system for comprehensive analysis of caspase 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 in view of the above circumstances, the present inventors have used an array in which a peptide recognizing caspase is immobilized on a metal-coated substrate, and further interaction between substances on the array. Has been found to be extremely useful for rapid analysis of the kinetics of various caspases.

That is, the present invention has the following configuration.
(1) An amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 and two or more peptides containing a biotin residue are immobilized on a gold surface. A peptide array for detecting caspase cleavage by surface plasmon resonance.
(2) The peptide array according to (1) for detecting caspase activity in a sample based on detection of cleavage of the peptide by surface plasmon resonance imaging.
(3) The peptide array according to (1) or (2), wherein the free end of a peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 is modified with biotin The peptide array according to any one of (1) to (3), which has a cysteine residue at the end of the array side of the peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 .
(5) The peptide array according to (4), wherein a spacer is inserted between an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 and a terminal cysteine residue.
(6) The peptide array according to (5), wherein the spacer is polyethylene glycol (PEG).
(7) The peptide array according to (5), wherein the spacer contains a chemical structure represented by formula (I) or (II).

(In the formula, n is 1 to 10, preferably 2 to 3. m represents 1 to 3.)
(8) A peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 is used, using a heterobifunctional cross-linking agent having a succinimide (NHS) group or a succinimide sulfate group and a maleimide (MAL) group, The peptide array according to any one of (1) to (7), which is immobilized on a surface.
(9) After performing a caspase reaction on a substrate using the peptide array according to any one of (1) to (8), a caspase is generated by a signal of surface plasmon resonance obtained by acting avidin or streptavidin. A method for detecting activity.
(10) The method for detecting caspase activity according to (9), wherein blocking is performed before performing the caspase reaction on the substrate.

  A measurement system for detecting caspase activity in an 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 novel drug discovery screening such as caspase inhibitors.

  The present invention is characterized in that exhaustive detection of peptide hydrolysis patterns by caspases in On-chip is performed by interaction analysis using 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 specification, “the peptide is immobilized on the gold surface” means that the peptide serving as a caspase substrate may be directly immobilized on the gold surface, and an appropriate spacer, a water solution such as polyethylene glycol, etc. 1 or 2 such as a hetero-functional cross-linking agent containing an NHS group and a maleimide group, a substance containing an SH group for binding to a gold surface or a SH group for bonding to a gold surface (cysteine, a compound having a terminal thiol group, etc.) It may be immobilized on the gold surface via seeds or more. Further, the peptide may further have a biotin residue bonded thereto, and a bivalent linking group such as a peptide can be interposed between the biotin residue and the peptide serving as a caspase substrate.

In the present invention, an array in which a peptide substrate is immobilized on a substrate as described above is used. As the peptide containing an amino acid sequence capable of functioning as a substrate for various caspases, at least two, preferably three or more peptides containing the amino acid sequences of SEQ ID NOs: 1 to 5 are used. These peptides have been confirmed to be responsive to various caspases. By selecting some of these five types and combining them for array analysis, comprehensive analysis of caspase activity is possible. Is something that can be done. As a detection means in array analysis, the detection method by SPR is particularly advantageous from the viewpoint of quantitative reproducibility, for example, compared to a method such as fluorescence array analysis.
The responsiveness of each of the five types of caspases is shown in Table 1 below.

  Furthermore, in order to confirm the reliability regarding the measurement, it is preferable that the peptide serving as a negative control is also immobilized on the same substrate. When using a peptide serving as a negative control, it is preferable to use one in which one or several amino acid sequences in SEQ ID NOs: 1 to 5 are replaced with different amino acids. Preferably, the amino acid sequence is preferably substituted with an amino acid that does not greatly change the amount of charge. Moreover, as a site | part which adds an amino acid substitution, adding to the cleavage site | part by a caspase is preferable. 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 preferably about 4 to 20 residues, more preferably about 4 to 10 residues.

  The peptide used in the present invention is preferably modified with biotin at one end. When the biotinylated peptide is immobilized, the amount of binding by avidin, streptavidin (SA), etc. decreases as the caspase cleavage reaction proceeds, so the signal decreases when SPR measurement is performed. By confirming, it is possible to detect caspase activity. Furthermore, it is preferable to cause an anti-SA antibody to act in that the difference in the binding property of SA depending on the presence or absence of caspase activity becomes clearer. This method is also preferable in that the caspase can be easily accessed to the molecule to be immobilized.

  The method for immobilizing peptides in the peptide array of the present invention is not particularly limited, and is a method involving a functional group such as an amino group or a thiol group in a peptide sequence, or an affinity bond such as a His tag, GST tag, or MBP tag. The method of using is mentioned. Among these, the method of immobilizing a peptide 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 preferably added to 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.

  In the present invention, it is more preferable 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, 100-2000 are preferable and 400-1000 are more preferable. In addition to the hydrophilic compound, a spacer sequence having 2 to 10 amino acid residues, more preferably 2 to 6 amino acid residues may be further added. The type of amino acid residue constituting the spacer sequence is not particularly limited, but it is preferable to include an amino acid that is 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.

  Alternatively, a compound containing a chemical structure as shown in the following formula (I) or formula (II) may be inserted. In this case, it is not limited to the formula (I) or the formula (II), and those having a longer chain length with these chemical formulas as the basic skeleton may be used. For example, derivatives of 12-amino-3,6,9-trioxaundecanoic acid and 8-amino-3,6-dioxanooctanoic acid can be used.

(In the formula, n is 1 to 10, preferably 2 to 3. m represents 1 to 3.)
By adding such a spacer, the degree of freedom of the immobilized substrate peptide can be improved, and as a result, the access to the immobilized peptide of caspase becomes easier, so that the efficiency of the effect of the caspase to be received is further increased. It becomes possible to raise. The position where the spacer is inserted is not particularly limited, but is preferably closer to the immobilization site. The method for synthesizing the peptide into which the hydrophilic compound is inserted is not particularly limited, and can be easily obtained by using, for example, a commercially available compound modified with a protecting group for peptide synthesis. The kind of the protecting group is not particularly limited, and general groups such as an Fmoc group and a tBoc group are used.

  In the present invention, when a peptide is immobilized via a thiol group, a heterobifunctional cross-linking agent having a succinimide (NHS) group or a sulfated succinimide group and a maleimide (MAL) group is introduced after introducing an amino group to the surface in advance. It is particularly preferable to use it. As such a heterobifunctional type crosslinking agent, a low molecular weight one may be used, or a hydrophilic polymer such as PEG having both ends modified with NHS groups and MAL groups may be used. is there. Any of these may be used, but a PEG derivative is preferably used. In this case, the molecular weight is preferably between 500 and 5,000, more preferably between 800 and 2,000.

  Examples of the low molecular weight heterobifunctional cross-linking agent include the compound Succinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (hereinafter referred to as SMCC) or the compound Succinoccinimidyl 4 represented by the formula (II). -[N-maleimidomethyl] cyclohexane-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 (III) or formula (IV) 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.

  In order to form a maleimide surface on a chip using the above-mentioned crosslinking agent, it is necessary to introduce a functional group having reactivity with a succinimide group or a succinimide sulfate group, specifically an amino group, in advance on the chip. . The means for introducing an amino group on the chip is not particularly limited. Examples thereof include a self-assembled surface method for aligning molecules on the substrate surface, a method of introducing using a reaction reagent, and a means for coating a substance having a functional group on a chip. Also included are means for introducing an amino group using a cross-linking agent starting from the functional group introduced on the surface.

  In the peptide array used in the present invention, the non-immobilized portion (background portion) of the peptide is preferably coated with a compound as shown in Formula (V). In formula (V), 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 measurement, it is preferable to use a means for suppressing the influence of nonspecific adsorption or the like by blocking, in order to obtain accurate measurement data. When using a maleimide surface, it is preferable to use a compound having a thiol group. For example, a derivative of PEG (polyethylene glycol) can be used. Alternatively, bovine serum albumin (BSA) or casein may be used. Moreover, the blocking agent which consists of phospholipids is also applicable.

  The present invention is directed to the detection of caspase activity on an array. Accordingly, when a solution containing or considered to contain caspase is allowed to act on the array obtained as described above, a compound that inhibits or is thought to inhibit the caspase activity must be allowed to coexist in the solution. become. The caspase to be subjected to profiling may be a commercially available reagent, but it is also possible to use a reagent that is already contained or presumed to be contained in a cell-derived disruption solution. For example, the caspase reagent or the caspase already contained in the cell lysate in the buffer or cell lysate (cell lysate) or a mixture of both can be directly applied to the array to effect the immobilized substrate.

  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 the response pattern of various caspase activities due to the drug. It is also possible to evaluate caspase activity inhibition in a system in which an inhibitor coexists. Furthermore, the peptide array of the present invention is also useful for screening for caspase promoters. Therefore, for example, screening of novel drugs such as various caspase inhibitors, and diagnosis of diseases such as cancer using caspases as markers can be performed.

  The reaction conditions vary depending on the type of caspase, but are usually fixed by reacting at a temperature of 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. The hydrolyzed peptide can be hydrolyzed. The reaction buffer is not particularly limited, but a neutral buffer is preferable. For example, a phosphate buffer, a HEPES buffer, a Tris-HCL buffer, a MOPS buffer, a MES buffer, or the like is used. The pH is preferably in the range of 6-8, and more preferably in the range of 6.5-7.5. Moreover, you may coexist the substance which assists and accelerates | stimulates enzyme activity in a reaction liquid as needed.

  The present invention can also be aimed at detecting inhibitory activity related to caspases on an array. In this case, when a solution containing or thought to contain caspase is allowed to act, a compound that inhibits or is thought to inhibit the caspase activity is allowed to coexist 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.

  A comprehensive analysis by profiling of caspase activity can be realized by conducting an on-chip reaction by the method of the present invention as described above. 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. NHS-dPEG12-MAL (manufactured by Quanta Biodesign) was dissolved in 20 mM phosphate buffer (150 mM NaCl; pH 7.2) at 1 mM and reacted with 8-AOT on the gold surface for 2 hours. 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.

  A peptide solution having a concentration of 100 μM (both dissolved in a phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at a concentration of 0.5 mM) was applied to the surface obtained as described above, and MultiSPRinter (registered trademark). Spotting was performed 10 nl at a time using an automatic spotter (manufactured by Toyobo). As shown in FIG. 2, the peptide to be immobilized has three cysteine residues and three linker molecules inserted, and the peptide uses the sequences shown in SEQ ID NOs: 1 to 9, and glycine (Gly) − A glycine (Gly) -serine (Ser) -lysine (Lys) sequence was added and the end was biotinized. As a linker, 8-amino-3,6-dioxanooctanoic acid was inserted. In this linker insertion, when the peptide is synthesized, Fmoc-mini-PEG (registered trademark; Fmoc-8-amino-3, 6-dioxanotactic acid manufactured by Petides International) is used to easily insert the linker. Can be obtained. 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 the surface on which the substrate peptide was immobilized was washed with a phosphate buffer, 1% BSA (PBS solution) was dropped on the chip and incubated at 37 ° C. for 3 hours for blocking.

[Example 2]
(Detection of caspase activity by SPR analysis)
Using the array obtained in Example 1 up to the blocking treatment, a reaction with caspase 5 was performed on the chip. 300 μl of the caspase 5 solution was dropped on the array and reacted at 37 ° C. for 16 hours. The composition of the caspase 5 solution was prepared by diluting caspase 5 (manufactured by MBL) with 50 mM HEPES (pH 7.2; 50 mM NaCl, 0.1% Chaps, 10 mM EDTA, 10 mM DTT) so as to be 0.1 unit / μl. Using.

  The array subjected to the caspase reaction was washed for 10 minutes each with ultrasonic treatment in the order of 0.05% Tween 20-containing TBS, 1% SDS-containing TBS, and milli-Q water. Thereafter, the array was set in an SPR imaging device (MultiSPRinter (registered trademark): manufactured by Toyobo Co., Ltd.), and PBS was allowed to flow through the flow cell as a running buffer at a rate of 100 μl / min. After confirming that the signal from SPR was stabilized, a 0.5 μg / ml concentration of streptavidin (SA; manufactured by Molecular Probes) solution was allowed to act for 20 minutes, and running buffer was fed for 3 minutes to perform washing. An anti-SA antibody solution (manufactured by Vector) having a concentration of 1 μg / ml was allowed to act for 20 minutes. Thereafter, the running buffer was again fed for 3 minutes for washing. All liquid feeding was performed at 0.1 ml / min. 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 graphing the signal value in the SPR sensorgram obtained for each probe is shown in FIG. As shown on the horizontal axis of the graph, P represents a peptide known to act as a substrate for each caspase, and N represents a negative control peptide for each caspase substrate. Regarding the caspase 5, 6 and 8 substrates, it can be presumed that the caspase 5 was cleaved by the action of caspase 5, since it was confirmed that the SA binding signal was reduced by the enzymatic reaction.

[Example 3]
On-chip reaction of caspase 6, caspase 8 and caspase 9 on the chip was performed in the same manner using the array obtained in Example 1 up to the blocking treatment. Caspase 6, caspase 8, and caspase 9 were all manufactured by MBL, and in the same manner as in Example 2, 50 mM HEPES (pH 7.2; 50 mM NaCl, 0.1% Chaps) was used so that each would be 0.1 unit / μl. 10 mM EDTA, 10 mM DTT), 300 μl was dropped on the array, and the reaction was carried out at 37 ° C. for 16 hours. Subsequent analysis by SPR was performed in the same manner as in Example 2.
The results are shown in FIGS. It can be seen that the binding signal of SA is reduced by the caspase action, and the tendency is different depending on the type of caspase.

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

It is a figure which shows the principle of SPR imaging. It is a figure which shows the structure of the fixed peptide probe used in Example 1. FIG. It is a figure which shows the result of having performed the SPR analysis regarding On-chip reaction by the caspase 5 implemented in Example 2. FIG. It is a figure which shows the result of having performed the SPR analysis regarding On-chip reaction by the caspase 6 implemented in Example 3. FIG. It is a figure which shows the result of having performed the SPR analysis regarding On-chip reaction by the caspase 8 implemented in Example 3. FIG. It is a figure which shows the result of having performed the SPR analysis regarding On-chip reaction by the caspase 9 implemented in Example 3. FIG.

Claims (10)

Cleavage of the peptide on a substrate by caspase, wherein two or more peptides containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 and a biotin residue are immobilized on a gold surface Peptide array for detecting the surface by surface plasmon resonance. The peptide array according to claim 1, for detecting caspase activity in a sample based on detection of cleavage of the peptide by surface plasmon resonance imaging. The peptide array according to claim 1 or 2, wherein a free end of a peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 is modified with biotin. The peptide array according to any one of claims 1 to 3, which has a cysteine residue at the end of the array side of the peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5. The peptide array according to claim 4, wherein a spacer is inserted between an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 and a terminal cysteine residue. The peptide array according to claim 5, wherein the spacer is polyethylene glycol (PEG). The peptide array according to claim 5, wherein the spacer contains a chemical structure represented by formula (I) or (II).

(In the formula, n is 1 to 10, preferably 2 to 3. m represents 1 to 3.) A peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 is immobilized on a gold surface using a heterobifunctional crosslinking agent having a succinimide (NHS) group or a succinimide sulfate group and a maleimide (MAL) group. The peptide array according to claim 1, wherein the peptide array is formed. A method for detecting caspase activity by a signal of surface plasmon resonance obtained by allowing avidin or streptavidin to act after performing a caspase reaction on a substrate using the peptide array according to any one of claims 1 to 8. . The method for detecting caspase activity according to claim 9, wherein blocking is performed before the caspase reaction is performed on the substrate.

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