Kinase activity measuring device and method having function of determining number of effective phosphorylation sites

Abstract

To provide a method for measuring kinase activity taking into account the number of phosphorylation sites.SOLUTION: A method for measuring kinase activity comprises: (a) associating the number of available phosphorylation sites with a substrate protein; (b) obtaining the amount of the substrate protein on a substrate; and (c) estimating kinase activity based on the number of available phosphorylation sites corresponding to the substrate protein on the substrate and the obtained amount of the substrate protein on the substrate.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The embodiments disclosed in the present specification and drawings relate to an apparatus and method for measuring kinase activity that has the function of determining the number of effective phosphorylation sites. [Background technology]

[0002] In cells, there are molecular mechanisms that transmit information to express various functions, and post-translational modifications of proteins, especially protein phosphorylation, play an important role in signal transduction. The intracellular signal transduction mechanism forms a complex network in which many molecules cooperate, and it is thought that abnormal signal transduction caused by activation due to mutations or overexpression of protein kinases, which are protein phosphorylation enzymes, is involved in many diseases.

[0003] In light of this background, techniques for measuring kinase activity have been developed and used. A common method for measuring kinase activity is to react a substrate protein or peptide with a kinase in a sample and measure the phosphorylated substrate to measure kinase activity. Known methods for detecting phosphorylated peptides or proteins include ELISA using anti-phosphorylated antibodies, mobility shift assay, peptide arrays, protein arrays, and LC-MS. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-50171 Summary of the Invention [Problem to be solved by the invention]

[0005] On the other hand, the number of phosphorylation sites contained in one molecule of a substrate protein or peptide varies depending on the type of substrate, and even when a fixed amount of substrate protein or peptide is reacted with a kinase, the number of phosphorylation sites that actually function as a substrate varies depending on the substrate type. Because the rate of phosphorylated amino acid production in a substrate protein or peptide is affected by the number of phosphorylation sites, conventional kinase activity measurements do not take into account the effect of differences in the number of phosphorylation sites.

[0006] One of the problems to be solved by the embodiments disclosed in this specification and the drawings is to enable measurement of kinase activity taking into account the number of phosphorylation sites. However, the problems to be solved by the embodiments disclosed in this specification and the drawings are not limited to the above problem. Problems corresponding to the effects of each configuration shown in the embodiments described below can also be positioned as other problems. [Means for solving the problem]

[0007] The kinase activity measurement method according to the embodiment includes the steps of: (a) associating the number of available phosphorylation sites with a substrate protein; (b) acquiring the amount of the substrate protein on a substrate; and (c) estimating kinase activity based on the number of available phosphorylation sites corresponding to the substrate protein on the substrate and the acquired amount of the substrate protein on the substrate.

[0008] The kinase activity measuring device according to the embodiment is an information processing device that includes an acquisition unit that acquires the amount of a substrate protein on a substrate, and an estimation unit that reads out the number of effective phosphorylation sites corresponding to the substrate protein on the substrate from a memory unit that stores the number of effective phosphorylation sites associated with the substrate protein, and estimates kinase activity based on the number of effective phosphorylation sites and the acquired amount of the substrate protein on the substrate. [Brief explanation of the drawings]

[0009] [Figure 1]FIG. 1 is a diagram showing an example of the flow of a kinase activity measurement method according to the first embodiment. [Figure 2] Figure 2 shows the straight line obtained by measuring the amount of phosphorylated amino acids and the amount of substrate protein by reacting a kinase with a substrate protein (BCAR1), and plotting the number of phosphorylated amino acids as a function of the amount of substrate protein. [Figure 3] Figure 3 shows the straight line obtained by measuring the amount of phosphorylated amino acids and the amount of substrate protein after reacting a kinase with a substrate protein (Abi1) and plotting the number of phosphorylated amino acids as a function of the amount of substrate protein. [Figure 4] Figure 4 shows the straight line obtained by measuring the amount of phosphorylated amino acids and the amount of substrate protein after reacting a kinase with a substrate protein (Src), and plotting the number of phosphorylated amino acids as a function of the amount of substrate protein. [Figure 5] Figure 5 shows the curve obtained when kinases with the same activity were reacted with substrate proteins with different numbers of sites, the number of phosphorylated amino acids and the amount of substrate protein were measured, and the number of phosphorylated amino acids was plotted on a graph as a function of the amount of substrate protein. [Figure 6] Figure 6 shows a curve obtained by correcting and normalizing the number of phosphorylated amino acids in the curve obtained in Figure 5 based on the number of phosphorylation sites obtained. [Figure 7] Figure 7 shows plots of the average phosphorylation reaction rate versus the number of phosphorylation sites obtained for BCAR1 and Src, and also shows the results of calculating Vmax-Km by fitting taking into account the initial number of phosphorylation sites. [Figure 8] Figure 8 shows a plot of the amount of phosphorylated amino acids against the number of phosphorylation sites obtained for BCAR1, and also shows the results of calculating Vmax-Km by fitting taking into account the decrease in the number of phosphorylation sites during the reaction. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the kinase activity measuring device and the kinase activity measuring method will be described in detail with reference to the drawings.

[0011] (First embodiment) The kinase activity measurement method according to the first embodiment comprises the steps of: (a) associating the number of available phosphorylation sites with a substrate protein; (b) acquiring the amount of the substrate protein on a substrate; and (c) estimating kinase activity based on the number of available phosphorylation sites corresponding to the substrate protein on the substrate and the acquired amount of the substrate protein on the substrate.

[0012] The method for measuring kinase activity according to the first embodiment comprises the step of (a) associating the number of effective phosphorylation sites with the substrate protein.

[0013] The "substrate protein" in the kinase activity measurement method according to the first embodiment refers to one or more proteins that are phosphorylated by a kinase whose activity is to be measured, and may be immobilized on a substrate or contained in a spot. The substrate protein can be appropriately determined depending on the purpose by the person who performs the kinase activity measurement method according to the first embodiment, and may be naturally occurring, synthetic, or recombinant.

[0014] The number of phosphorylation sites (sites that undergo phosphorylation reaction) in a substrate protein may vary depending on the type of substrate protein. In the method for measuring kinase activity according to the first embodiment, the number of phosphorylation sites per molecule of a specific substrate protein is defined as the number of effective phosphorylation sites corresponding to that specific substrate protein. The number of effective phosphorylation sites corresponding to a specific substrate protein is preferably set for each combination of substrate protein and kinase.

[0015] The method for obtaining the number of available phosphorylation sites corresponding to a specific substrate protein is not particularly limited, and the number may be obtained directly by calculation, for example, by plotting on a graph the amount of phosphorylated amino acids formed in a labeled phosphorylated substrate protein in a spot as a function of the amount of substrate protein in the spot and calculating the slope of the resulting line or curve, or by contacting the substrate protein with a kinase for a period that allows phosphorylation of all available phosphorylation sites in the substrate protein and calculating the number of available phosphorylation sites. The obtained data on the number of available phosphorylation sites corresponding to a specific substrate protein may be stored and, if necessary, may be used as the value for the number of available phosphorylation sites corresponding to a specific substrate protein in another measurement.

[0016] Alternatively, if there is a preset value for the number of effective phosphorylation sites for a specific substrate protein, that value may be used with reference. In this case, if the number of effective phosphorylation sites corresponding to a specific substrate protein is publicly known, that value may be used with reference, for example, a value identified using the PhosphoSitePlus online tool. In this case, it is preferable to use a value for a kinase of the same species as the kinase to be measured as the number of effective phosphorylation sites corresponding to a specific substrate protein, but a value for a kinase of a similar species to the kinase to be measured may also be used as the number of effective phosphorylation sites corresponding to a specific substrate protein.

[0017] The method for measuring kinase activity according to the first embodiment comprises the step (b) of acquiring the amount of substrate protein on the substrate.

[0018] The method for obtaining the amount of substrate protein is not particularly limited, and can be, for example, quantification by SPR (surface plasmon resonance), quantification based on electrochemical properties (e.g., potential, current value, impedance, capacitance, etc.), quantification based on the abundance distribution of elements obtained by X-ray spectroscopy, etc., quantification using AFM, quantification based on signal information derived from a labeling substance introduced into the substrate, etc. Furthermore, if the amount of substrate protein is known, it may be used.

[0019] The "substrate" in the kinase activity measurement method according to the first embodiment is not particularly limited, and examples thereof include two-dimensional plates, polymer gels, fibers and fiber sheets, beads, and rods. The surface of the "substrate" may be smooth, or may have a micro / nano structure such as a porous structure or fibers. The "substrate" is preferably a two-dimensional plate, and examples of two-dimensional plates include plate-like substrates such as glass slides and cover glasses, and well substrates such as array plates. The "substrate" is more preferably a well substrate such as an array plate. An array plate has spots containing a substrate and is used for comprehensive analysis of samples; it is also called a microchip, microarray, protein chip, DNA chip, etc.

[0020] A "spot" in the kinase activity measurement method according to the first embodiment refers to a certain enclosed area on a substrate. The shape of the "spot" is not particularly limited and may be, for example, a square, rectangle, circle, oval, etc., but is preferably a circle. A substrate protein may be contained in the spot, and in this case, the contained substrate protein may be immobilized on the substrate.

[0021] The kinase activity measurement method according to the first embodiment includes (c) a step of estimating kinase activity based on the number of effective phosphorylation sites corresponding to the substrate protein on the substrate and the amount of the substrate protein obtained on the substrate.

[0022] In the kinase activity measurement method according to the first embodiment, the "sample" containing the kinase to be measured can be determined appropriately by the person performing the method depending on the purpose, and anything containing a kinase can be used as a sample. Samples include biologically derived substances, extracts from biological organisms, blood, blood-derived substances, food, food-derived substances, natural products, substances derived from natural products, and culture medium-derived substances. Depending on the purpose and procedure, the sample may be pretreated as appropriate, or a reagent may be added to the sample beforehand. The sample may be in the form of a gas, solid, or liquid, but may be diluted, suspended, or extracted in water, physiological saline, a buffer solution, or other solution and used in liquid form. The sample may contain preservatives and other additives. Furthermore, a reagent may be added to the sample depending on the purpose.

[0023] The method for measuring kinase activity according to the first embodiment may be a method for measuring the kinase activity of a kinase contained in a sample using a substrate having spots containing a substrate.

[0024] Fig. 1 is a diagram showing an example of the flow of a kinase activity measurement method according to the first embodiment. The kinase activity measurement method shown in Fig. 1 includes the steps of performing a phosphorylation reaction between a kinase and a substrate protein to obtain a phosphorylated substrate protein (phosphorylation reaction step), further labeling the phosphorylated substrate protein to obtain a labeled phosphorylated substrate protein (labeling step), measuring the amount of phosphorylated amino acids formed in the labeled phosphorylated substrate protein (and the amount of the substrate protein, if necessary) ((substrate protein amount and) phosphorylated amino acid amount measurement step), acquiring the amount of substrate protein and the number of effective phosphorylation sites corresponding to the substrate protein (substrate protein amount and) number of effective phosphorylation sites acquisition step), estimating the value of the kinase activity of the kinase based on the amount of substrate protein, the number of effective phosphorylation sites corresponding to the substrate protein, and the amount of phosphorylated amino acids formed in the labeled phosphorylated substrate protein (kinase activity estimation step), and outputting the kinase activity of the kinase based on the estimated value (kinase activity output step).

[0025] The kinase activity measurement method shown in Figure 1 includes a step of carrying out a phosphorylation reaction between a kinase and a substrate protein to obtain a phosphorylated substrate protein. This step may be a step of bringing a sample into contact with a spot on a substrate to carry out a phosphorylation reaction between the kinase contained in the sample and the substrate protein contained in the spot, thereby obtaining a phosphorylated substrate protein.

[0026] The kinase activity measurement method shown in FIG. 1 includes a step of further labeling the phosphorylated substrate protein to obtain a labeled phosphorylated substrate protein. Further labeling the phosphorylated substrate protein means introducing the labeling substance into the phosphorylated substrate protein via a phosphorylation site recognition substance. The phosphorylation site recognition substance is a substance that specifically recognizes the phosphorylated site in a substrate protein that has been phosphorylated by a kinase. The phosphorylation site recognition substance is not particularly limited, and examples include anti-phosphorylated amino acid antibodies. When a labeling substance is not bound to the phosphorylation site recognition substance, the labeling substance may be introduced via a secondary antibody or a specific reaction including a biotin-avidin reaction.

[0027] Examples of labeling substances include radioactive substances, enzymes, capture molecules, fluorescent substances, luminescent substances, metal particles, etc., and preferably those that are optically detectable. Examples of such labeling substances include fluorescent substances, chemiluminescent substances, phosphorescent substances, dyes, gold nanoparticles, fluorescent particles, enzymes for enzymatic chemiluminescence or color reactions, and microparticles that absorb specific wavelengths. Labeling substances may also include antibodies, ligands, or other binding moieties. The binding or introduction of labeling substances can be accomplished by binding the labeling substance via hydrophobic interactions, electrostatic interactions, van der Waals interactions, hydrogen bonds, or covalent bonds, or by introduction during synthesis, or by other known labeling methods.

[0028] The method for measuring kinase activity shown in Figure 1 includes a step of measuring the amount of phosphorylated amino acids in a labeled phosphorylated substrate protein, and, if necessary, the amount of the substrate protein. The amounts of the phosphorylated amino acids in the labeled phosphorylated substrate protein and the substrate protein in the spot may be measured.

[0029] The amount of substrate protein can be measured by known methods, including, for example, quantification by SPR (surface plasmon resonance) method, quantification based on electrochemical properties (e.g., potential, current value, impedance, capacitance, etc.), quantification based on element distribution obtained by X-ray spectroscopy, etc., quantification using AFM, quantification based on signal information derived from a labeling substance introduced into the substrate, etc. Furthermore, if the amount of substrate protein is known, it may be used.

[0030] The amount of phosphorylated amino acid formed in the labeled phosphorylated substrate protein is quantified based on signal information derived from the labeling substance. Information on the amount of labeled phosphorylated substrate protein is obtained based on the signal intensity, i.e., it becomes possible to quantitate the amount of phosphorylated amino acid formed in the labeled phosphorylated substrate protein. Such a signal is preferably optically detectable, and examples thereof include light intensity information and spectral information from the spot.

[0031] When the labeling substance is a fluorescent substance, the optical system can be any optical system capable of exciting the fluorescent substance and detecting the fluorescence. An excitation light source can be used to excite the fluorescent substance, and examples of such excitation light sources include a laser light source, a light-emitting diode, an LED, a mercury arc, and a tungsten halogen lamp. A CCD camera, a photodiode, or the like may be used for detection. The optical system may include a filter as appropriate, and irradiate or detect light of a limited wavelength. The optical system may also include a lens. The optical system may be a scanning or non-scanning type. Specifically, a confocal optical unit can be used as the optical system.

[0032] When chemiluminescence, dyes, or the like are used as the labeling substance, an excitation light source is not necessary.

[0033] The method for measuring kinase activity shown in Figure 1 includes a step of obtaining the amount of a substrate protein and the number of available phosphorylation sites corresponding to the substrate protein. The amount of the substrate protein and the number of available phosphorylation sites corresponding to the substrate protein may be measured in a spot.

[0034] The method for measuring kinase activity shown in Figure 1 includes a step of estimating the value of the kinase activity of the kinase based on the amount of a substrate protein, the number of available phosphorylation sites corresponding to the substrate protein, and the amount of phosphorylated amino acids generated in the labeled phosphorylated substrate protein.

[0035] The kinase activity value in this step is estimated by correcting the amount of phosphorylated amino acids generated in the labeled phosphorylated substrate protein, measured based on the number of effective phosphorylation sites corresponding to the substrate protein. For example, this may be done by simply normalizing the measured amount of phosphorylated amino acids generated in the labeled phosphorylated substrate protein by the determined number of effective phosphorylation sites corresponding to the substrate protein, but preferably, this is done taking into account the decrease in the number of effective phosphorylation sites corresponding to the substrate protein as the phosphorylation reaction progresses.

[0036] Kinase activity values ​​that take into account the decrease in the number of available phosphorylation sites corresponding to the substrate protein as the phosphorylation reaction progresses can be estimated, for example, by: (i) obtaining the amount of substrate protein at each point on the spot at the beginning of the reaction and the amount of phosphorylated amino acids produced in the phosphorylated substrate protein after a specified reaction time has elapsed; (ii) converting the amount of substrate protein at the beginning of the reaction into the number of available phosphorylation sites corresponding to the substrate protein; (iii) assuming certain Km and Vmax, and causing the production of phosphorylated amino acids and the decrease in the number of available phosphorylation sites corresponding to the substrate protein after a unit time has elapsed based on the phosphorylation production rate determined based on the number of available phosphorylation sites corresponding to the substrate protein at the beginning of the reaction; (iv) determining the phosphorylation production rate in the next unit time based on the number of available phosphorylation sites corresponding to the substrate protein after the decrease; (v) repeating (iii) and (iv) to obtain an estimate of the relationship between the initial substrate protein amount and the amount of phosphorylated amino acids produced at the end of a specified reaction time; and (vi) converging Vmax and Km so that the error between the estimate obtained in (v) and the actual measurement result is minimized. According to a method for measuring kinase activity that includes this step, it is possible to eliminate bias due to differences in the number of effective phosphorylation sites corresponding to the substrate protein, and to more accurately measure the activity of the kinase to be measured.

[0037] The method for measuring kinase activity shown in FIG. 1 includes a step of outputting the kinase activity of the kinase based on the estimated value.

[0038] (Second embodiment) The kinase activity measuring device according to the second embodiment is an information processing device comprising: an acquisition unit that acquires the amount of a substrate protein on a substrate; and an estimation unit that reads the number of effective phosphorylation sites corresponding to the substrate protein on the substrate from a storage unit that stores the number of effective phosphorylation sites associated with the substrate protein, and estimates kinase activity based on the number of effective phosphorylation sites and the acquired amount of the substrate protein on the substrate. The storage unit may be further provided in the information processing device, or may be connected to the information processing device via a network.

[0039] The meanings and indications of the terms "substrate protein," "substrate," "number of effective phosphorylation sites," etc. used to describe the second embodiment are the same as those used to describe the first embodiment.

[0040] In the acquisition section of the kinase activity measuring device according to the second embodiment, the amount of substrate protein may actually be measured and the measurement result may be acquired as the amount of substrate protein on the substrate, or if the amount of substrate protein is publicly known, that value may be acquired as the amount of substrate protein on the substrate.

[0041] According to at least one of the embodiments described above, it is possible to measure kinase activity taking into account the number of phosphorylation sites.

[0042] Although several embodiments have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, modifications, and combinations of embodiments can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims. [Example]

[0043] The present invention will be described in detail based on the following examples, but the present invention is not limited to these examples. Contents are expressed in mass % unless otherwise specified.

[0044] Example 1. Estimation of the number of phosphorylation sites in each substrate protein We selected three substrate proteins: GST (glutathione-S-transferase)-tagged BCAR1, GST-tagged Abi1, and GST-tagged Src. We performed an example of estimating the number of phosphorylation sites on these substrate proteins for kinases contained in cell extracts of the human leukemia cell line K562. While there are three amino acids commonly phosphorylated: serine, threonine, and tyrosine, we used tyrosine phosphorylation to estimate the number of phosphorylation sites on each substrate.

[0045] For each substrate protein, a dilution series of spotting solutions was prepared so that the protein concentration ratios were 1, 2, 4, and 8. Four spots of each concentration of the spotting solution were dropped onto a glutathione (GSH)-coated glass slide prepared by the method described in a non-patent document (Tadashi Manabe et al., "IGF2 Autocrine-Mediated IGF1R Activation Is a Clinically Relevant Mechanism of Osimertinib Resistance in Lung Cancer", Mol Cancer Res. 2020 Apr;18(4):549-559.), yielding an array plate with solid-phase spots (approximately 100 μm in diameter) of GST-fused BCAR1, Abi1, and Src. In addition, an excess amount (approximately 100 μg) of cell extract from the human leukemia cell line K562 was mixed with a kinase reaction solution (25 mM Tris-HCl, 5 mM β-glycerophosphate, 0.1 mM Na3VO4, 10 mM MgCl2, 1 mM ATP, and 2 mM DTT) and contacted with the immobilized spots of each protein. This was then incubated at 30°C for a sufficient period (approximately 2 hours) to induce phosphorylation at all phosphorylation sites of each substrate protein that were active against the kinase contained in the K562 cell extract. The reaction was then stopped by contacting the immobilized spots with a reaction stop solution (50 mM EDTA, 10 mM HEPES-NaOH [pH 7.4], 150 mM NaCl, and 0.05% [v / v] Tween 20) and incubating at 30°C for 5 minutes. After washing the array plate with TBST, the primary antibody reaction solution (mouse anti-phosphotyrosine A cocktail of rabbit anti-GST antibodies and rabbit anti-GST antibodies was added and incubated at 30°C for 1 hour.After washing the array plate with TBST, a secondary antibody solution (a cocktail of goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 790 (Invitrogen) and goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 680 (Invitrogen)) was added and incubated at 30°C for 1 hour. The phosphorylated tyrosine in the immobilized Src spots was labeled with Alexa Fluor 790, and the GST tag in Src was labeled with Alexa Fluor 680. Fluorescence images of the labeled array plate were captured using a microarray scanner with confocal optics. Alexa Fluor 790 was detected using a 780 nm excitation laser, and Alexa Fluor 680 was detected using a 670 nm excitation laser. For each substrate protein, the fluorescence intensity (F780-B780, where F780 is the average fluorescence intensity per spot and B780 is the average fluorescence intensity in the area outside the spot) indicating the amount of phosphorylated tyrosine per spot was plotted as a function of the fluorescence intensity (F670-B670, where F670 is the average fluorescence intensity per spot and B670 is the average fluorescence intensity in the area outside the spot) indicating the amount of substrate per spot (Figures 2, 3, and 4). Under the conditions used in this experiment, all available phosphorylation sites were phosphorylated, so the spot intensity, indicating the amount of phosphorylated tyrosine for each protein, was linear with a constant slope relative to the spot intensity, indicating the amount of substrate. This slope indicates the amount of phosphorylated tyrosine obtained per unit amount of substrate protein. Furthermore, under conditions where the phosphorylation reaction is saturated, the amount of phosphorylated tyrosine indicates the number of available phosphorylation sites. Therefore, by comparing the slopes among BCAR1, Abi1, and Src, the ratio of the number of available phosphorylation sites was obtained. The slope ratio for BCAR1:Abi1:Src was: TIFF2025181505000001.tif5166 This was the effective phosphorylation site number ratio for BCAR1, Abi1, and Src when phosphorylated with K562 cell extract. To calculate the number of phosphorylation sites from the phosphorylation site number ratio, the number of phosphorylation sites reported for BCAR1 was determined by referencing the publicly known database, phosphositeplus. phosphositeplus contains information on the phosphorylation sites for each substrate protein, obtained primarily by LC-MS and other techniques for various samples. In this example, among the tyrosine residues for which phosphorylation detection has been reported for BCAR1, tyrosine residues with 50 or more reported cases were selected as the maximum number of potential phosphorylation sites, and 12 phosphorylation sites were extracted. Considering that the slope ratio of BCAR1:Abi1:Src is 5:0.5:1 and the number of phosphorylation sites is an integer, possible ratios of the number of phosphorylation sites include 10:1:2 = 20:2:4 = 30:3:6. However, since the maximum number of potential phosphorylation sites is 12 and the number of effective phosphorylation sites does not exceed 12, we estimated that the number of effective phosphorylation sites for the three kinases contained in the K562 cell extract (BCAR1, Abi1, and Src) is 10, 1, and 2, respectively.

[0046] In measuring the activity of the kinase group contained in K562, the number of available phosphorylation sites was estimated by multiplying the amount of substrate measured for each substrate spot by the number of available phosphorylation sites obtained above.

[0047] Example 2. Normalization of phosphorylated amino acid levels by the number of phosphorylation sites To determine kinase activity, a reaction solution containing a kinase is contacted with an array plate having spots of substrate proteins to induce a phosphorylation reaction, and the amount of substrate protein and phosphorylated amino acid per spot is measured. An example is shown in which the amount of phosphorylated amino acid is normalized by the number of phosphorylation sites.

[0048] An array plate having spots of substrate proteins was prepared, phosphorylation reaction by kinase was induced, labeling was performed, and the amounts of substrate proteins and phosphorylated amino acids were measured using the same procedures as in Example 1. However, since determining kinase activity required that only a portion of the phosphorylation sites be phosphorylated (i.e., not all phosphorylation sites were phosphorylated and phosphorylation was not saturated), the reaction conditions were controlled by reducing the amount of cell extract or shortening the phosphorylation reaction time if all phosphorylation sites were phosphorylated.

[0049] The substrate amount per spot was multiplied by the number of phosphorylation sites on that substrate protein to convert it to the number of phosphorylation sites per spot. The amount of phosphorylated amino acids per spot was also considered to be the average phosphorylation reaction rate over the set phosphorylation reaction time. Under phosphorylation reaction conditions with a small number of phosphorylation sites, the phosphorylation reaction rate is rate-limited by the number of phosphorylation sites, making it difficult to simply use the amount of phosphorylated amino acids per spot as an indicator of kinase activity. Therefore, the amount of phosphorylated amino acids per spot was normalized by dividing it by the number of phosphorylation sites per spot. This normalization allowed us to correct for the effect of the number of phosphorylation sites on the reaction rate, and then compare the magnitude of kinase activity.

[0050] An example of correction is shown in Figure 5. Here, as an example of phosphorylation reaction, the Michaelis-Menten equation is used.

number

[0051] Example 3. Estimation of Vmax-Km by fitting using the Michaelis-Menten equation To determine kinase activity, a reaction solution containing a kinase is contacted with an array plate having spots of substrate proteins to induce a phosphorylation reaction, and the amount of substrate protein and phosphorylated amino acid per spot is measured. In this example, the maximum phosphorylation reaction rate Vmax and the Michaelis constant Km are determined by fitting the distribution of the number of phosphorylation sites and the amount of phosphorylated amino acids at the initial stage of the reaction to the Michaelis-Menten equation.

[0052] An array plate having spots of substrate proteins was prepared, phosphorylation reaction by kinase was induced, labeling was performed, and the amounts of substrate proteins and phosphorylated amino acids were measured using the same procedures as in Example 1. However, since determining kinase activity requires that only a portion of the phosphorylation sites be phosphorylated (i.e., not all phosphorylation sites are phosphorylated and phosphorylation is not saturated), if all phosphorylation sites are phosphorylated, the reaction conditions were controlled by reducing the amount of cell extract or shortening the phosphorylation reaction time.

[0053] Based on the measurement results of the amounts of substrate protein and phosphorylated amino acids, the distribution of the amount of phosphorylated amino acids versus the amount of substrate protein was plotted two-dimensionally. To perform Michaelis-Menten fitting, it was necessary to plot the amount of phosphorylated amino acids versus different amounts of substrate protein. This was obtained by preparing spots using spotting solutions with different substrate protein concentrations and plotting the amount of phosphorylated amino acids per spot versus the amount of substrate protein per spot, as in Example 1. Alternatively, when variations in the amount of immobilized substrate protein were observed at various points within a single spot prepared using a spotting solution with a certain substrate protein concentration, the amount of substrate protein versus the amount of phosphorylated amino acid at each point was plotted.

[0054] For the plots obtained, the amount of substrate protein was multiplied by the number of phosphorylation sites of that substrate protein, and the amount of phosphorylated amino acids was divided by the phosphorylation reaction time to convert it into the average phosphorylated amino acid production rate, and the distribution of the phosphorylated amino acid production rate against the number of phosphorylation sites was obtained. The obtained distribution was then applied to the Michaelis-Menten equation:

number

[0055] For BCAR1 and SRC spots, 10 μg of K562 cell extract was used for 120-minute phosphorylation reactions. The substrate protein and phosphorylated amino acid amounts were determined by plotting the fluorescence intensity, which indicates the amount of substrate protein and phosphorylated amino acid at each point within the spot. The number of phosphorylated amino acid sites was set to 10 and 2 (values ​​determined in Example 1), respectively. The relationship between the number of phosphorylated amino acid sites and the average reaction rate, calculated by dividing the amount of phosphorylated amino acid by the phosphorylation reaction time (120 minutes), was then plotted. The results of fitting were performed using the method of Example 3. The phosphorylation reaction conditions were as follows: the amount of lysate was reduced compared to Example 1. This indicated a nonlinear correlation between the number of phosphorylated sites and the amount of phosphorylated amino acid, suggesting that only a portion of the phosphorylation sites were phosphorylated (i.e., phosphorylation was not saturated). Fitting yielded Km = 133,560 and Vmax = 350 ( / min) for BCAR1, and Km = 9,916 and Vmax = 201 ( / min) for Src. By taking into account the difference in the number of phosphorylation sites, the difference in affinity between BCAR1 and Src could be expressed as a difference in Km, suggesting that Src has a smaller Km and a higher affinity for the kinases contained in the K562 cell extract. Note that, because there is a background fluorescence intensity (F780-B780) that indicates the amount of phosphorylated amino acids, the average phosphorylation reaction rate was set to a background of 7.1 for BCAR1 and 46.7 for Src.

[0056] In this example, by applying the Michaelis-Menten equation to the distribution of phosphorylated amino acid amounts relative to the number of phosphorylation sites, it is believed that Vmax can be estimated more accurately as kinase activity than in Example 2. The Km determined by fitting in this example indicated the number of phosphorylation sites at the initial stage of the reaction at which the average phosphorylation reaction rate could be half the maximum reaction rate Vmax after 120 minutes of phosphorylation reaction.

[0057] Example 4. Estimation of Vmax-Km using the Michaelis-Menten equation, taking into account the decrease in the number of phosphorylation sites during the reaction To determine kinase activity, a reaction solution containing a kinase is contacted with an array plate having spots of substrate protein to induce a phosphorylation reaction, and the amount of substrate protein and phosphorylated amino acid per spot is measured. In this example, the maximum phosphorylation reaction rate Vmax and the Michaelis constant Km are determined by fitting to the Michaelis-Menten equation, taking into account the number of phosphorylation sites at the beginning of the reaction and the decrease in the number of unreacted phosphorylation sites during the reaction.

[0058] An array plate having spots of substrate proteins was prepared, phosphorylation reaction by kinase was induced, labeling was performed, and the amounts of substrate proteins and phosphorylated amino acids were measured using the same procedures as in Example 1. However, since determining kinase activity requires that only a portion of the phosphorylation sites be phosphorylated (i.e., not all phosphorylation sites are phosphorylated and phosphorylation is not saturated), if all phosphorylation sites are phosphorylated, the reaction conditions were controlled by reducing the amount of cell extract or shortening the phosphorylation reaction time.

[0059] As in Example 3, a plot showing the distribution of phosphorylated amino acid amounts versus substrate protein amount was obtained. Furthermore, a plot showing the distribution of phosphorylated amino acid amounts versus the number of phosphorylation sites was obtained by multiplying the substrate protein amount by the number of phosphorylation sites for that substrate protein species. Here, the amount of substrate protein was determined using the fluorescence intensity upon labeling with a GST tag fused to the substrate protein as an indicator. Therefore, the number of phosphorylation sites obtained represents the number present at the beginning of the reaction, and the number of unreacted phosphorylation sites decreases as the phosphorylation reaction progresses. Taking this into consideration, a Michaelis-Menten fitting was performed. Assuming a certain Km and Vmax, the phosphorylated amino acid production rate and the decrease in the number of unreacted phosphorylation sites after a unit time elapsed were determined for the number of unreacted phosphorylation sites measured and calculated at the beginning of the reaction. Furthermore, the phosphorylation production rate for the next unit time was determined based on the number of phosphorylation sites after the decrease. This procedure was repeated until the phosphorylation reaction time was reached, and the correspondence relationship between the number of phosphorylation sites and the amount of phosphorylated amino acid amount was estimated. Vmax and Km were obtained by converging them so as to minimize the error between the estimated correspondence relationship and the distribution of the amount of phosphorylated amino acids and the number of phosphorylation sites actually measured. In this example, compared to Example 3, the phosphorylation reaction rate per unit time was calculated taking into account the effect of a decrease in the reaction rate due to a decrease in the number of unreacted substrate sites, eliminating bias due to differences in the number of available phosphorylation sites on the substrate and enabling more accurate measurement of the activity of the target kinase.

[0060] The graph showing the relationship between the number of phosphorylated amino acid sites and the amount of phosphorylated amino acids in the BCAR1 spots obtained in Example 3 was fitted using the method of Example 4, and the results are shown in Figure 8. As shown in Figure 8, Vmax = 349 ( / min) and Km = 115,724 were obtained. The Km value is smaller than in Example 3, but this suggests that by performing fitting taking into account the decrease in the number of unreacted phosphorylation sites, it is possible to avoid overestimating Km, and a Km closer to the true value could be estimated.

[0061] The Km and Vmax values ​​obtained in Examples 2 to 4 are based on the measured values ​​of fluorescence brightness and are therefore in arbitrary units. However, it is also possible to convert the units by, for example, creating a calibration curve based on the relationship between the known amount of phosphorylated amino acid and fluorescence brightness, and the known number of phosphorylation sites and fluorescence brightness.

Claims

1. (a) associating the number of available phosphorylation sites with a substrate protein; (b) obtaining the amount of substrate protein on the substrate; and (c) estimating kinase activity based on the number of available phosphorylation sites corresponding to the substrate protein on the substrate and the amount of the substrate protein obtained; 10. A method for measuring kinase activity comprising:

2. The method of claim 1, wherein step (a) comprises contacting the substrate protein with a kinase for a period of time that allows phosphorylation of all available phosphorylation sites in the substrate protein, calculating the number of available phosphorylation sites, and correlating the calculated number of available phosphorylation sites with the substrate protein.

3. The method of claim 1, wherein step (a) comprises a step of referring to a predetermined value as the number of available phosphorylation sites for a substrate protein and associating the referenced number of available phosphorylation sites with the substrate protein.

4. The method according to any one of claims 1 to 3, wherein the number of effective phosphorylation sites for a substrate protein is associated with each combination of a substrate protein and a kinase.

5. an acquisition unit for acquiring the amount of substrate protein on the substrate; an estimation unit that reads out the number of effective phosphorylation sites corresponding to a substrate protein on the substrate from a storage unit that stores the number of effective phosphorylation sites associated with a substrate protein, and estimates kinase activity based on the number of effective phosphorylation sites and the amount of the substrate protein on the substrate; An information processing device comprising:

6. The information processing device according to claim 5 , further comprising the storage unit.

7. The information processing device according to claim 5 , wherein the storage unit is connected to the information processing device via a network.

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