KR100451594B1 - Determination Method of Protein Phosphorylation Reaction by Enzyme Activity - Google Patents

Abstract

The present invention provides a method for analyzing protein phosphorylation by enzymes using a capillary electrophoresis device and a matrix assisted laser desorption / ionization mass spectrometer.

The method for analyzing protein phosphorylation by enzyme of the present invention comprises the steps of setting the conditions of the capillary electrophoresis device for measuring the phosphorylation reaction of MBP peptide by ERK, determining the MBP peptide that phosphorylation reaction by ERK is effectively performed; Detecting the phosphorylation reaction of MBP peptide of ERK using a set capillary electrophoresis device and a matrix assisted laser desorption / ionization mass spectrometer.

Intracellular response assays using the capillary electrophoresis device or matrix assisted laser desorption / ionization mass spectrometry of the present invention can greatly reduce the economic cost by reducing the cost, time and effort for the study of in vivo signal transduction systems. It is considered

Description

Determination Method of Protein Phosphorylation Reaction by Enzyme Activity

The present invention provides a method for analyzing protein phosphorylation by enzymes using a capillary electrophoresis device and a matrix assisted laser desorption / ionization mass spectrometer.

The present invention relates to the development of protein phosphorylation reaction analysis method of enzymes in the signaling mechanism inside the cell, specifically, the extracellular signal-regulated kinase by the activation of external cellular stimulation (Extracellular Signal-Regulated Kinase, 'ERK' Phosphorylation reaction of the protein by using a capillary electrophoresis device.

With the recent increase of various pollutants and harmful substances to human body, it is urgent to understand the causes of various diseases, mental disorders caused by cranial nerve disorders, allergies, language disorders, and physical development disorders. As the basic research for the prevention and treatment of such diseases and the development of drugs, it is essential to study the fundamental signaling system that occurs in the body. At present, the developed countries are investing a lot of investment and research personnel in the fundamental research for the development of therapies and drugs, but the research in our country is still insufficient. Therefore, the problem of understanding the underlying mechanism is urgent, and the economic ripple effect such as the development of new medicine based on such research is enormous.

Protein phosphorylation is considered the basic mechanism of cellular response regulation in all eukaryotic organisms. In many pathways, cells use phosphorylation reactions on various proteins, for example as signaling mechanisms to regulate metabolism, protein synthesis, gene expression, growth, division, morphological changes, and the like. When a phosphorylation reaction occurs to a protein of interest by an appropriate protein kinase, the proteins or enzymes that act as regulators are labeled or protected at the site of activation to target other regulators.

In the present invention, in the study of intracellular signaling mechanisms related to cellular responsiveness to various external stimuli, new analytical methods that complement the difficulties and problems of existing analytical methods have been applied and applied to a broader study.

The study of signaling mechanisms is accomplished by understanding the function of each signaling protein. In general, the study of these functions consists of comparing the similarities to the genes already identified by the isolation of the genes, but the expected outcome of the proteins. Sometimes it consists of a method of confirming. Moreover, in the absence of possible probes, isolation of genes according to protein function is often the only way.

Significant technological advances have been made in identifying signaling proteins that interact with each other in many cells using these techniques.However, there are many limitations in the study of their function by separating and purifying the proteins present in cells. This followed. Recently, attempts have been made to introduce microanalysis into protein function analysis to overcome these limitations.

In studying the function of intracellular signaling proteins, it is very important to study the activation of proteins to external stimuli. Until recently, most of the methods for confirming the activation of signaling proteins were mostly using isotopically labeled substrates or energy sources. Cellular reactants were identified by gel electrophoresis or confirmed using antibodies. The method has been used mainly.

The most common method was to attach ³² P or [γ- ³² P] organophosphate directly to cultured cells [RL Stariha, S. Kikuchi, YL Siow, SL Pelech, M. Kim, SU Kim, J. Neurochem. 68 (1997) 945]. These methods have facilitated the study of intracellular signaling mechanisms, but there are many problems associated with the use of radioisotopes, such as high cost or time investment and concern about environmental pollution. For this reason, studies of these signaling mechanisms in complete tissues, whole plants, animals or humans have been extremely limited.

In order to overcome this limitation, a method using an antibody has been developed, but an antibody that recognizes phosphorylated tyrosine residues suffers from a lack of sensitivity and specificity as well as a somewhat qualitative measurement [H. Arad-Dann, U. Beller, R. Haimovitch, Y. Gavrieli, S. A. Ben-Sasson, J. Histochem. Cytochem. 41 (1993) 513]. In addition, antibodies for recognizing phosphorylated serine residues and phosphorylated threonine residues are also commercially available, but these also have the same problem.

In particular, the phosphorylation reaction of the protein by the enzyme, there was a risk and expensive cost to use isotope, but this problem was solved through the development of a new method using a capillary electrophoresis device.

Recently used capillary electrophoresis and matrix assisted laser desorption / ionization mass spectrometry are breakthrough analysis systems for detecting a wide range of molecules, from small organic molecules to large molecules such as nucleic acids or proteins. Due to the advantage of relatively simple analysis within, many studies have been made as a method for overcoming the problems of the existing analytical methods.

The analysis method using capillary electrophoresis device is a method that separates according to the speed difference in the capillary of charged sample by applying high voltage to both ends of the capillary tube and is currently very useful with high resolution and short analysis time. Laser desorption / ionization mass spectrometry is a mass spectrometry that measures the molecular weight of a sample by ionizing the desorbed sample using a laser and a matrix.

In the case of capillary electrophoresis, various separation modes have been recently developed to facilitate the separation of various materials, and the advantages of cost reduction, adequate detection sensitivity, excellent selectivity, and fast analysis time have been applied in the fields of medical analysis and biotechnology. Application is being attempted.

Matrix-assisted laser desorption / ionization mass spectrometers are also unaffected by the ability to accurately measure the mass of materials in the mass range up to hundreds of thousands of daltons, the detection limits of picomolars, buffer solutions used in biological samples, and salts. As an analytical method having the advantage that it can be directly analyzed without purification, it is easy to analyze a small amount of material using the same.

However, in the case of studying the phosphorylation reaction of the substrate by the enzyme using a conventional capillary electrophoresis device, because the sensitivity of the sample is low, a special detector such as an LIF detector has to be used to improve the sensitivity. In this case, since the derivatization process is required for this, there is a problem that the experimental process is complicated and the analysis takes a very long time. In addition, it has been very difficult to analyze the phosphorylation of threonine residues by capillary electrophoresis.

Accordingly, the object of the present invention is to solve the problems of the conventional enzyme assay as described above, and the analysis process is simple and within a short time using a capillary electrophoresis device and a matrix assisted laser desorption / ionization mass spectrometer having many advantages. The present invention provides a method for analyzing protein phosphorylation that can be analyzed and detects phosphorylation of threonine residues.

1 shows peptides synthesized around the active site of myelin basic protein (MBP), a substrate that is phosphorylated by extracellular signal-regulated kinase (hereinafter abbreviated as 'ERK'). Table showing amino acid sequence, molecular weight and pI value.

FIG. 2A is a graph showing the peak pattern of KNIVTPRTPPPSQGK peptide when using 150 mM Tris-Phosphate buffer solution at pH 2.8 to which different concentrations of acetonitrile were added at 0%, 5% and 10%.

FIG. 2B is a graph showing the peak pattern of VPRTPGGRR peptide when using 150 mM Tris-Phosphate buffer solution at pH 2.8 to which different concentrations of acetonitrile were added at 0%, 5% and 10%.

FIG. 2C is a graph showing the peak pattern of APRTPGGRR peptide when using 150 mM Tris-Phosphate buffer solution at pH 2.8 with different concentrations of acetonitrile added at 0%, 5%, 10%.

Figure 3a is a graph showing the results of analysis of the phosphorylation reaction by ERK of MBP peptide having the KNIVTPRTPPPSQGK amino acid sequence using a capillary electrophoresis device.

Figure 3b is a graph showing the results of analyzing the phosphorylation reaction by ERK of MBP peptide having a VPRTPGGRR amino acid sequence using a capillary electrophoresis device.

Figure 3c is a graph showing the results of analysis of the phosphorylation reaction by ERK of MBP peptide having the APRTPGGRR amino acid sequence using a capillary electrophoresis device.

Figure 4 is a graph showing the observation of the phosphorylation reaction of MBP peptide while varying the amount of ERK to 0, 10, 20, 30 and 40 U / ml.

5 is a graph showing the results of analyzing the phosphorylation of MBP peptides by ERK according to the presence of ATP using a capillary electrophoresis apparatus.

FIG. 6A is a graph showing the detection results of MBP peptides without phosphorylation reaction by ERK using a matrix assisted laser desorption / ionization mass spectrometer. FIG.

Figure 6b is a graph showing the detection results of MBP peptides phosphorylated by ERK using a matrix assisted laser desorption / ionization mass spectrometer.

The present inventors set the conditions of capillary electrophoresis apparatus capable of analyzing the phosphorylation reaction of MBP peptide by activated ERK, and after detecting the phosphorylation reaction of protein under the set conditions, the matrix assisted laser desorption / ionization mass spectrometer It was found that the object can be achieved by confirming the phosphorylation reaction through.

Accordingly, the present invention provides a method for analyzing protein phosphorylation by enzymes using a capillary electrophoresis device and a matrix assisted laser desorption / ionization mass spectrometer.

More specifically, the method for analyzing protein phosphorylation by enzyme of the present invention is to set the conditions of the capillary electrophoresis apparatus for measuring the phosphorylation reaction of MBP peptide by ERK, MBP peptide that phosphorylation by ERK is effectively performed Determining and detecting phosphorylation of MBP peptides in ERK using a set capillary electrophoresis device and a matrix assisted laser desorption / ionization mass spectrometer.

First of all, the conditions for capillary electrophoresis to measure phosphorylation of MBP peptides by ERK include the internal diameter and length of the capillary, the determination of the detector, the composition and pH of the buffer solution of the capillary electrophoresis, and the buffering. Determining the concentration of the organic solvent added to the solution. These steps are necessary for detecting MBP peptides from a mixture of various substances included in order for phosphorylation of MBP peptides by ERK to occur.

Determination of phosphorylation reaction conditions of MBP peptide by ERK consists of three MBP peptides synthesized with different amino acid sequences around the active site by ERK, ie, -PRT ⁹⁷ P-, of the substrate MBP peptide. Detecting the degree of phosphorylation according to the difference. This step is necessary to select MBP peptides that can detect the phosphorylation reaction more effectively than in capillary electrophoresis. After this step, the phosphorylation reaction is detected using a capillary electrophoresis apparatus at an optimal state using the set conditions.

Finally, the phosphorylation reaction of MBP peptide of ERK detected using capillary electrophoresis is performed using matrix assisted laser desorption / ionization mass spectrometry. This step includes a matrix selection step, conditions for the detection of phosphorylation in a matrix assisted laser desorption / ionization mass spectrometer.

The present invention is simpler because the derivatization process by using a special detector such as a LIF detector is not required, compared with the case of studying the phosphorylation reaction of the substrate by an enzyme using a conventional capillary electrophoresis device. Detecting phosphorylation of threonine residues, which are short in assay time and generally difficult to detect, can provide a cornerstone for easier analysis methods in the future.

Hereinafter, the configuration and operation of the present invention will be described in detail with reference to the following examples, but the scope of the present invention is not limited only to the following examples.

In the examples below, ERK was purchased from New England Biolabs, 25 mM beta glycol phosphate, 1 mM sodium olovavanadate, 5'-deoxyadenosine triphosphate (ATP), ethylene glycol-bis (Aminoethyl ether) N, N, N ', N'-tetraacetic acid (EGTA), dithioltrethiol (DTT) and magnesium chloride were purchased from Sigma Chemical and MBP peptides were obtained from Anigen It synthesize | combined and used. The capillary electrophoresis system used is Waters Quanta 4000 CE System, manufactured by Water, and MALDI-TOF-MS is HP G2025A, manufactured by Hewlett-Packard.

Example 1 Condition Setting of Capillary Electrophoresis Device for Analyzing Phosphorylation of Proteins

Analytical conditions of the capillary electrophoresis device were determined based on the separation of unphosphorylated MBP peptides and phosphorylated MBP peptides within a short assay time with high sensitivity. The sample used here is an MBP peptide having the amino acid sequence of KNIVTPRTPPPSQGK and a phosphorylated MBP peptide (KNIVTPRT-pPPPSQGK) having one phosphate group attached thereto. These were all synthesized by Anigen, and 5 ul of 20 mM tris-hydrochloride of pH 7.2 containing 25 mM beta glycol phosphate, 1 mM sodium olobanadate, 5 mM EGTA, and 1 mM DTT and 75 mM MgCl. 5 ul of a ₂ -500 uM ATP mixture and an enzyme dilution solution (50 mM tris-hydrochloride solution at pH 7.5 containing 100 mM sodium chloride, 0.1 mM disodium EDTA, 1 mM DTT, 0.01% Bril35 and 50% glycerol) 5 ul 5 ul was added at a concentration of 100 ug / ml each. In addition to the KNIVTPRTPPPSQGK peptide, the peptides APRTPGGRR and VPRTPGGRR shown in FIG. 1 were also used in the same manner.

(Step 1) Determination of inner diameter and length of capillary and detection wavelength

In capillary electrophoresis, the inner diameter and length of the capillary are the most basic setting conditions and have a great influence on the material analysis. In general, the inner diameters that are frequently used are 50 μm, 75 μm, and 100 μm. As the inner diameter increases, the sensitivity increases, but the resolution decreases. In this example, 75 μm was selected in order to select a condition having good sensitivity and separation of unphosphorylated MBP and phosphorylated MBP peptide.

The length of the capillary tube varies depending on the model of the capillary electrophoresis apparatus, and the minimum length of 35 cm is used in the Waters Quanta 4000 CE system used in this example. In the case of materials with poor resolution and poor separation, materials with a slow moving speed require a long capillary length, but the longer the length, the longer the analysis time. Therefore, the minimum length that can be separated and analyzed is generally selected. In this example, since both the unphosphorylated MBP peptide and the phosphorylated MBP peptide were isolated and detected at 35 cm, 35 cm was selected as the appropriate capillary length.

Therefore, in this Example, 75 [mu] m and 35 cm were set to the inner diameter and length of the most suitable capillary to separate the phosphorylated MBP peptide from the phosphorylated MBP peptide.

In this example, a UV detector was used and selected at a wavelength of 185 nm. In general, when using a UV detector, a wavelength of 245 nm or 214 nm is used. This is because there is an advantage that the sensitivity of the sample is very high at low wavelengths, such as 185 nm, but this is because it is difficult to detect and analyze all the other substances of low concentration contained in the sample or buffer solution.

In this embodiment, as shown in each figure, this problem was solved by detecting all other substances 1-2 minutes before the unphosphorylated MBP peptide was detected. Therefore, in this example, a UV detector with a wavelength of 185 nm was selected for more sensitive analysis.

(Step 2) Determination of the composition and pH of the buffer solution of the capillary electrophoresis apparatus

In Figure 1, it can be seen that the pI value of the KNIVTPRTPPPSQGK peptide is very large. If the pI value of the peptide is large, it will be well adsorbed to the capillary tube. Therefore, a low pH of pH 3 or lower, or a high pH buffer solution above the peptide should be used.

Since the pI value of the KNIVTPRTPPPSQGK peptide is very high at 11.17, separation of the silanol groups in the capillary inner wall dissociates well when using a buffer solution of a higher pH. Therefore, a low pH buffer solution must be used.

First, the composition of the buffer solution was selected as the most popular phosphate buffer solution at low pH, and each sample was detected at concentrations of 50 mM, 100 mM, 150 mM, and 200 mM to determine the concentration. As a result, the higher the concentration of the phosphate buffer solution, the sensitivity was improved, but at 200 mM, the stabilization time was too long (more than 1 hour) was selected 150 mM phosphate buffer solution.

Thereafter, separation of the KNIVTPRTPPPSQGK peptide and its phosphorylated peptide was observed in buffer solutions at low pH, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0. As a result, the higher the pH, the higher the sensitivity and the longer the analysis time. In the case of sensitivity, pH 3.0 was the best, but there was a concern of adsorption and the analysis time was longer than 10 minutes, so the pH 2.8 150 mM phosphate buffer solution was determined as the buffer solution of the capillary electrophoresis device.

In this condition, acetonitrile was added to reduce the risk of adsorption and improve sensitivity. As the concentration of acetonitrile was increased to 0%, 5%, 10%, 20%, 30%, and 40%, the higher the acetonitrile concentration, the higher the sensitivity. However, MBP peptide was added when more than 30% acetonitrile was added. The sample of was gradually overlapped with other peaks.

Figures 2a, 2b and 2c show the peak behavior of each MBP peptide at concentrations of 0%, 5% and 10% of acetonitrile, with the other peaks almost preceding at 10% of acetonitrile for Figure 2b. You can see that it is adjacent to. Therefore, in this example, it was finally decided to use a 150 mM phosphate buffer solution at pH 2.8 with 5% acetonitrile added.

Example 2 Determination of Conditions for Phosphorylation of Protein

Phosphorylation of MBP peptides by ERK is accomplished by mixing ERK with a mixture of magnesium and ATP and MBP peptide. At this time, the phosphorylated group of ATP is transferred to the MBP peptide by ERK to produce the phosphorylated MBP peptide.

At this time, the enzyme reaction may occur differently due to the structural difference of the different amino acid sequences of the protein. In this example, the difference was observed using MBP peptides having amino acid sequences of KNIVTPRTPPPSQGK, APRTPGGRR and VPRTPGGRR.

Each MBP peptide was mixed with 5 ul of 20 mM tris-hydrochloride at pH 7.2 containing 25 mM beta glycol phosphate, 1 mM sodium olovavanadate, 5 mM EGTA, 1 mM DTT and 75 mM MgCl ₂ -500 uM ATP 5 ul and enzyme dilution solution (50 mM tris-hydrochloride solution at pH 7.5 containing 100 mM sodium chloride, 0.1 mM disodium EDTA, 1 mM DTT, 0.01% Bril35 and 50% glycerol) to 4 ul and 1 ul of ERK, respectively. 5 ul was added at a concentration of 100 ug / ml. Here, the reaction was stirred in a water bath at 30 ° C. for 30 minutes and analyzed using a capillary electrophoresis device.

3A, 3B and 3C show the results, showing a reduced aspect of MBP peptides. When comparing the respective figures, the sensitivity of the VPRTPGGRR peptide of Figure 3b shows the best, but the amount of reduction of the VPRTPGGRR peptide by phosphorylation reaction was also smaller than the APRTPGGRR peptide of 3c. Therefore, in this example, it was found that the most phosphorylation reaction occurs in the APRTPGGRR peptide of Figure 3c.

Example 3 Analysis of Phosphorylation of Proteins by Capillary Electrophoresis

Phosphorylation was observed by varying the amount of ERK at 0, 10, 20, 30, 40 U / ml using APRTPGGRR peptide. The APRTPGGRR peptide contains 5 ul of 20 mM tris-hydrochloride at pH 7.2 containing 25 mM beta glycol phosphate, 1 mM sodium olovavanadate, 5 mM EGTA, 1 mM DTT and 5 ul of 75 mM MgCl ₂ -500 uM ATP mixture. And an enzyme dilution solution (50 mM tris-hydrochloride solution at pH 7.5 comprising 100 mM sodium chloride, 0.1 mM disodium EDTA, 1 mM DTT, 0.01% Bril35 and 50% glycerol) and 0, 10, 20, 30, 40 5 ul of URK was added to 5 ul of ERK at a concentration of 100 ug / ml, respectively. Here, the reaction was stirred in a water bath at 30 ° C. for 30 minutes and analyzed using a capillary electrophoresis device.

This result is shown in FIG. As shown in Figure 4, as the amount of ERK increased to 0, 10, 20, 30, 40 U / ml it was observed that the phosphorylation reaction was also increased and also the degree gradually decreased. This is because the reaction efficiency gradually decreases as the reaction reaches saturation.

5A and 5B are graphs showing the results of detection by the capillary electrophoresis apparatus when the phosphorylation reaction of the APRTPGGRR peptide by ERK did not occur and when the phosphorylation reaction occurred.

FIG. 5A shows an APRTPGGRR peptide containing 10 mM ul 20 mM tris-hydrochloride at pH 7.2 containing 25 mM beta glycol phosphate, 1 mM sodium olsovadate, 5 mM EGTA, 1 mM DTT (100 mM sodium chloride, 50 ulm tris-hydrochloride solution of pH 7.5 containing 0.1 mM disodium EDTA, 1 mM DTT, 0.01% Bril35 and 50% glycerol) 3 ul, 2 ul of ERK, 5 ul each at a concentration of 100 ug / ml The reaction was carried out in a water bath at 30 ° C. for 30 minutes and then analyzed using a capillary electrophoresis device. As a result, no phosphorylation reaction occurred because no ATP-MgCl ₂ mixture was added to the reaction, and no phosphorylated peptide of the APRTPGGRR peptide was observed.

However, in the case of FIG. 5B, the APRTPGGRR peptide was prepared using 5 ul and 75 mM MgCl ₂ at 20 mM tris-hydrochloride at pH 7.2 containing 25 mM beta glycol phosphate, 1 mM sodium olovavanadate, 5 mM EGTA, 1 mM DTT. 5 ul of -500 uM ATP mixture, 3 ul of enzyme dilution solution (pH 7.5 mM solution of pH 7.5 containing 100 mM sodium chloride, 0.1 mM disodium EDTA, 1 mM DTT, 0.01% Bril35 and 50% glycerol) 5 ul was added to 2 ul of ERK at a concentration of 100 ug / ml and the reaction was carried out by stirring in a water bath at 30 ° C. for 30 minutes.

That is, unlike FIG. 5A, an ATP-MgCl ₂ mixture was added to the reaction, and as a result, the phosphorylated peptide of the APRTPGGRR peptide was confirmed at 10.4 minutes. At this time, the APRTPGGRR peptide also decreased, but the concentration of the APRTPGGRR peptide was higher than the phosphorylated peptide of the generated APRTPGGRR peptide, which was not seen in FIGS. 5A and 5B. In this example, the conversion of APRTPGGRR peptide to phosphorylated APRTPGGRR peptide was 14.7% according to the phosphorylation of MBP peptide by ERK.

Example 4 Identification Using Matrix Assisted Laser Desorption / Ionization Mass Spectrometry

The phosphorylation of MBP peptides by ERK was analyzed using capillary electrophoresis and observed using a matrix-assisted laser desorption / ionization mass spectrometer.

When analyzing materials in a matrix assisted laser desorption / ionization mass spectrometer, the choice of the matrix is the most important. In this example, a-CHCA matrix and DHB matrix were used for the analysis of MBP peptide. The a-CHCA matrix was prepared by dissolving acetonitrile and methanol in a solution of 5: 3: 2 at a concentration of 33 mM, and the DHB matrix was 100 mM in a 1: 1 solution of acetonitrile and water. It was prepared by dissolving to a concentration of.

The sample used was the sample used in Example 3 (FIG. 5). After taking 5 ul of each of these samples, 5 ul of a-CHCA matrix or DHB matrix was mixed thereto, and then 1 ul of the amount was placed on the probe and crystallized while standing at room temperature. The signal was analyzed by placing the sample in a matrix assisted laser desorption / ionization mass spectrometer and ionizing the sample into a linear TOF tube using a laser. As a result, the peak sensitivity was higher when the a-CHCA matrix was used than when the DHB matrix was analyzed, and the a-CHCA matrix was selected as the most appropriate matrix.

6a and 6b show the results of experiments using the DHB matrix. The molecular weight of the APRTPGGRR peptide was 967.2 Da, and the peaks with one more hydrogen in FIGS. 6A and 6B were detected at 968.2 Da, respectively. In FIG. 6B, phosphorylated APRTPGGRR peptide, the product of phosphorylation of APRTPGGRR peptide by ERK, was found at 1048.2 Da. This is exactly the same as the molecular weight of one phosphate group with a difference of 80 Da. Thus, the peak of the phosphorylated product was observed by the exact difference in molecular weight, it was confirmed that the phosphorylation reaction of the MBP peptide by the ERK occurs.

Intracellular reaction assays using the capillary electrophoresis device or matrix assisted laser desorption / ionization mass spectrometry of the present invention are simple and easy to analyze and can detect the phosphorylation of threonine residues in the body. It is thought to be very helpful in reducing the economic cost by bringing down the cost, time and effort for researching the signaling system.

Claims (2)

Determining the optimal pH and concentration of the buffer solution for measuring phosphorylation of myelin basic protein (MBP) peptides by extracellular signal-regulated kinase (ERK), Determining an optimal concentration of the compound which prevents adsorption of the peptide on the inner wall of the capillary and increases the detection sensitivity, and adding it to the buffer solution, Determining MBP peptides on which the phosphorylation reaction by ERK is effectively performed under the same conditions, centering on -PRTP- which is an active site by ERK of MBP peptide, Detecting phosphorylation of the MBP peptide by ERK using a capillary electrophoresis apparatus using the buffer solution and Further confirming the phosphorylation detected in the step using a matrix assisted laser desorption / ionization mass spectrometer A method for analyzing phosphorylation of threonine residues of protein MBP by enzyme ERK. The method of claim 1, wherein detecting phosphorylation of the MBP peptide by ERK is performed using a UV detector installed in a capillary electrophoresis device.