An in vitro detection method for CMT3 protein activity based on accurate quantification of SAM and SAH

Abstract

The invention discloses an in vitro detection method for CMT3 protein activity based on accurate quantification of SAM and SAH, which belongs to the field of biotechnology, and the steps are: synthesizing a double-stranded DNA fragment substrate rich in CHG sequence and a histone peptide according to the characteristics of protein activity; constructing an in vitro methyl transfer reaction system; incubating the reaction system at 37°C to perform an in vitro experiment of CMT3 methyl transfer activity; using ultra-high performance liquid chromatography and triple quadrupole tandem time-of-flight mass spectrometry in positive ion multiple reaction monitoring mode to accurately quantify SAM and S-adenosylhomocysteine ​​in the reaction system, and comparing and accurately calculating with a control group containing only TA substrate to show the methyl transfer activity of CMT3 protein. The invention provides a non-radioactive, simple and fast experimental means, which provides a new idea for a rapid and accurate quantitative method of a series of methyltransferase activities that depend on SAM methyl donors such as CMT3 protein.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an in vitro detection method for CMT3 protein activity based on accurate quantification of SAM and SAH. Background Art

[0002] Chromomethylase 3 (CMT3) can covalently bind a methyl group on a methyl donor S-adenosyl-L-methionine (SAM) to the carbon 5 site of the cytosine base in the DNA molecule to perform DNA methylation modification (5-methyl-cytosine, 5mC), and simultaneously produce a molecule of S-adenosyl homocysteine ​​(SAH) as a byproduct. 5m C type DNA methylation is currently the most widely studied and important epigenetic mark (DNA methylation in this application refers to this type of methylation). CMT3 is mainly responsible for maintaining mCHG type methylation in the genome (where H represents non-G bases of A, T or C) and is a key protein in the DNA methylation maintenance pathway. Generally, CMT3 needs to bind to the dimethylated Lysine 9 of H3 Histone (H3K9me2) to target DNA sites to complete methyl transfer activity and achieve specific DNA methylation.

[0003] Currently, the in vitro detection of CMT3 methyl transfer activity usually uses the isotope tracer method. The article “Dual Binding of Chromomethylase Domains to H3K9me2-Containing Nucleosomes Directs DNA Methylation in Plants” by Du et al. in CELL 2012-9 Vol.151 discloses a method for detecting the in vitro activity of Arabidopsis CMT3, which includes the following steps: 1) preparing an in vitro reaction mixture system, comprising the following components: 2.5 μL of tritiated 3H-SAM labeled with radioactive isotope tritium (3H), 3-5 μmol H3K9me2 peptide, 125 ng DNA substrate, 50 ng CMT3 protein, a buffer system comprising 50 mM Tris–HCl, 5 mM NaCl and 5% glycerol, a total reaction volume of 25 μL, and a pH value adjusted to 8.0; 2) performing an in vitro CMT3 activity reaction at room temperature for 60 min, placing the reaction mixture in a dry ice / ethanol bath and adding 2 μL of proteinase K; 4) adding 10 5) The filter paper was dried and immersed in EcoliteMP liquid scintillation mixture, and the radioactive isotope level in the filter paper was quantitatively measured using a Beckman LS1701 scintillation counter; 6) The results of the experimental group with added CMT3 purified protein were compared with those of the control group without added CMT3 purified protein in terms of radioactivity level (Counts per minute, CPM), and a significance analysis was performed to qualitatively reflect the activity level of CMT3.

[0004] A similar radioisotope-based in vitro activity detection method for ZMET2 (the homologous protein of CMT3 in corn) is also disclosed in the article “Mechanistic basis for maintenance of CHG DNA methylation in plants” by Fang et al. in NATURE COMMUNICATIONS 2022-7 Vol. 13, which includes the following steps: 1) preparing an in vitro methyl transfer reaction system: the reaction system contains 0.25 µM ZMET2, 1.0 µM histone peptide (H3K9me2), 2.0 µM DNA substrate, 0.56 µM 3H-SAM, 1.96 µM SAM, 50 mM Tris-HCl buffer (pH 8.0), 100 mM NaCl, 0.05% β-mercaptoethanol, 5% glycerol and 200 µg / mL bovine serum albumin (BSA); 2) the reaction mixture is incubated at 37°C for 20 min, and then 5 µL of pre-cooled SAM (10 mM) to terminate the reaction; 3) After the reaction, 8 µL of the mixture was loaded onto Hybond N + nylon membrane and dried at room temperature; 4) the membrane was then washed three times with 0.2 M ammonium bicarbonate (pH 8.2), once with deionized water, and once with 95% ethanol; 5) the membrane was dried and transferred to 4 mL of liquid scintillation buffer, and the radioactivity level of tritium in the membrane was measured using a Beckman LS6500 counter; 6) the group without H3K9me2 was used as the control group for data comparison and significance analysis, and the radioactivity level result (CPM) was used as the unit to qualitatively reflect the activity level of ZMET2.

[0005] However, the above existing methods have the disadvantages of high laboratory qualification level requirements, scarcity of liquid scintillation related instruments and equipment, difficulty in obtaining radioactive reagents, cumbersome institutional reagent approval process, lack of laboratory personnel with radioactive related operation qualifications, potential radiation hazards in the execution process, etc. At the same time, it is impossible to define the protease activity on a unit basis and accurately quantify the enzyme activity accordingly. Therefore, it is urgent to develop new in vitro activity detection methods for a series of methyltransferases such as CMT3 that rely on SAM methyl donors. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide a highly sensitive, highly accurate, safe and efficient in vitro detection method for CMT3 protein activity, which avoids the use of isotope-labeled radioactive compounds. The method can quickly and accurately quantify SAM and SAH in the system after the reaction to accurately calculate the activities of a series of methyltransferases such as CMT3 that rely on SAM methyl donors, solving the problems of the difficulty of executing such protease activity detection methods, long experimental cycles, and the inability to accurately define enzyme activity.

[0007] The present invention is specifically implemented through the following technical solutions:

[0008] An in vitro detection method for CMT3 protein activity based on accurate quantification of SAM and SAH comprises the following steps:

[0009] (1) Constructing an in vitro methyl transfer reaction system;

[0010] (2) Perform CMT3 methyl transfer in vitro reaction;

[0011] (3) Accurately quantify SAM and SAH after the reaction;

[0012] (4) Analyze and compare the quantitative results and calculate the in vitro methyl transfer activity.

[0013] Preferably, the total volume of the in vitro methyl transfer reaction system in step (1) is 40-60 μL, the system pH is 7.5-8.5, and contains 1-5 μM CMT3 purified protein, 1-5 μM double-stranded DNA substrate, 1-5 μM H3K9me2 histone peptide, 1-3 μM SAM, 30-60 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 5-20 mM sodium chloride (NaCl), and 3-6% glycerol.

[0014] Preferably, the in vitro reaction conditions of CMT3 methyl transfer in step (2) are incubation at 37°C for 30 to 120 min; the reaction is stopped by repeated freezing and thawing with liquid nitrogen for 2 to 5 times and adding 5 to 10 μL of proteinase K, followed by heating at 55°C for 5 to 15 min.

[0015] Preferably, the precise quantification of SAM and SAH in the system after the reaction in step (3) is performed by UPLC-qTOF-MS / MS.

[0016] Preferably, the chromatographic conditions of step (3) are as follows: the injection volume of the reaction solution is 5 μL; a CSH C18 chromatographic column (AcquityUPLC CSH C18 Column, 130 Å, 1.7 µm, 2.1 mm × 100 mm, Waters Corporation, Milford, USA) is used; the mobile phase A is 10 mM ammonium acetate and 0.2% acetic acid, and the mobile phase B is methanol; and a gradient elution is performed for 8 min (total flow rate is 0.3 mL / min, 1 min, 2% B; 5 min, 75% B; 6 min, 2% B).

[0017] Preferably, the mass spectrometry conditions of step (3) are as follows: UPLC-qTOF-MS / MS uses positive ion MRM mode to preset the mass-to-charge ratio information of SAM (Precursor ion=399.15, Fragment ion=250.1) and SAH (Precursor ion=385.13, Fragment ion=136.06); curtain gas is 35 psi, ion source gas 1 and gas 2 are both 55 psi, ion source temperature is 600°C, and ion spray voltage is 5500 volts.

[0018] Preferably, during the quantitative calculation process of step (4), the quantitative results of the SAM and SAH experimental group concentrations are compared with the control group results, and combined with the significance analysis, it is determined whether there is a significant difference from the control group (p < 0.05). If there is a significant difference, the enzyme activity is accurately calculated based on the definition of the SAM reduction or SAH increase caused by CMT3 activity per unit time.

[0019] Further preferably, according to the characteristics of CMT3 protein activity, the DNA substrate in the reaction system of step (1) should include a control group containing only TA bases and an experimental group rich in CHG bases, where H = A / T / G bases. If targeting a series of methyltransferases that rely on SAM methyl donors, the experimental group DNA substrate should be additionally designed.

[0020] More preferably, the reaction system in step (1) should be prepared in a 200 μL PCR tube. After preparation, the system should be flicked and gently mixed.

[0021] Further preferably, the volume of the reaction system in step (1) must be greater than 40 μL to ensure that the tip of the injection needle can be inserted into the liquid surface for sampling during automatic injection in step (3).

[0022] More preferably, after the reaction in step (2) is completed, all the reaction system is pipetted into a liquid injection bottle loaded with a microplastic liner and transferred completely into the liner.

[0023] More preferably, step (3) is performed by automatic injection using an Acquity UPLC-qTOF-MS / MS (Waters Corporation, Milford, USA) device and a liquid injection bottle loaded with a micro plastic liner through an automatic injector.

[0024] More preferably, a certain concentration gradient of SAM and SAH standard solutions should be prepared and dissolved in 30-60 mM Tris-HCl, 5-20 mM NaCl, and 3-6% glycerol, and steps (2) and (3) should be performed simultaneously to obtain a standard curve.

[0025] Further preferably, when there is a significant difference in SAM or SAH concentration between the experimental group and the control group in step (4), the CMT3 activity should be quantitatively calculated according to the following formula:

[0026]

[0027] in, U SAM The activity definition is based on the reduction of SAM content, which indicates the reduction of reactant SAM per unit time due to CMT3 activity, with the unit of pmol SAM / h; U SAH represents the activity definition based on the increase in SAH content, indicating the increase in product SAH per unit time due to CMT3 activity, with the unit of pmol SAH / h; further, c 对照 is the concentration of SAM or SAH in the control group (μM); c 实验 is the concentration of SAM or SAH in the experimental group (μM); v is the total volume of the reaction system (μL); t is the reaction time.

[0028] The present invention is the first to detect the in vitro methyl transfer activity of methyltransferases such as CMT3 by accurately quantifying the reduction of SAM and the increase of SAH by UPLC-qTOF-MS / MS, avoiding the use of isotope-labeled radioactive compounds, and has the advantages of safety, simplicity, sensitivity, environmental protection, high efficiency, and accuracy, specifically:

[0029] 1) The present invention avoids the use of isotope-labeled radioactive compounds in the prior art methods, thereby eliminating the need for laboratory radioactivity qualification level requirements, the scarcity of liquid scintillation-related instruments and equipment, the difficulty of obtaining radioactive reagents, the cumbersomeness of institutional reagent approval procedures, radioactivity-related operation qualification training, and whether there are potential radiation hazards in the execution process. It has the advantages of safety, environmental protection, and simplicity.

[0030] 2) The present invention uses UPLC-qTOF-MS / MS technology to achieve accurate quantification of SAM and SAH. It can directly and accurately calculate the activity of methyltransferases such as CMT3 that use SAM as a methyl donor based on the classic definition of enzyme activity, rather than the traditional method of indirectly qualitatively judging enzyme activity based on numerical comparison of CPM. It has the advantages of accuracy and strong universality of results.

[0031] 3) Steps (1) to (3) in the present invention are preparations for activity detection, and the UPLC-qTOF-MS / MS technology in step (4) is used as the actual detection step. The elution time for a single sample is only 8 minutes. Compared with the prior art, the present invention has the advantages of being simple, rapid, and efficient.

[0032] 4) Based on the MRM mode in UPLC-qTOF-MS / MS, the present invention presets the molecular information of SAM and SAH. Compared with the traditional isotope tracer method, the present invention has the advantages of strong specificity and high sensitivity.

[0033] 5) The present invention uses a reaction system containing only TA bases as the control group. Compared with the existing method in which no CMT3 purified protein and no H3K9me2 peptide are added as the control group, the difference between the components of the experimental group is minimal, which can play a better control effect and serve as a more appropriate background value, thereby obtaining more accurate quantitative results of CMT3 enzyme activity, and has the advantages of accuracy and reliable results. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the [M+H] in the UPLC-qTOF-MS / MS detection results after the reaction of the reaction system in Example 1 + Primary mass spectrum and corresponding secondary mass spectrum (where the reaction system contains octoploid cultivated strawberry ( Fragaria × ananassa Duch) CMT3 purified protein (FaCMT3), different double-stranded DNA substrates, H3K9me2 histone peptides, SAM, Tris-HCl, NaCl, glycerol);

[0035] Figure 2 The concentration results of SAM and SAH after the methyl transfer reaction in the reaction system in Example 1. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution.

[0037] Example 1

[0038] This embodiment adopts the method disclosed in the present invention, and calculates the enzyme activity by detecting the changes in the contents of the reactant SAM and the product SAH in the methyl transfer reaction. Specifically, one methyl group of one methyl donor SAM molecule is transferred to the 5th carbon of one cytosine molecule in the DNA double helix, thereby forming a methylated cytosine. At the same time, one molecule of SAH and one proton are generated due to the loss of one methyl group by SAM. The present invention uses UPLC-qTOF-MS / MS technology to accurately quantify the concentration changes of SAM and SAH, and can accurately calculate the in vitro methyl transfer activity value of CMT3. In order to detect the in vitro methyl transfer activity of the purified CMT3 protein (FaCMT3) of octoploid cultivated strawberry (Fragaria ×ananassa Duch), and to explore the preference of strawberry FaCMT3 for different types of DNA substrates, the implementation steps are as follows:

[0039] (1) Construct the reaction system: add 2 μM FaCMT3 purified protein, 4 μM double-stranded DNA substrate, 4 μM histone peptide, 2.5 μM SAM, 50 mM Tris-HCl, 10 mM NaCl, 5% glycerol, and ultrapure water to 60 μL.

[0040] The DNA single-strand base sequence and peptide amino acid sequence are shown in Table 1 (X = 5mC). The DNA substrate is formed by annealing the sense strand and the antisense strand to form a double strand for the reaction, where "CG" = a DNA substrate rich in CG type sequences but without methylation in both strands, "hmCG" = a hemimethylated DNA substrate rich in CG type sequences and all CG type sequences in one strand are methylated; the same applies to "CHG / hmCHG" and "CHH / hmCHH".

[0041] Table 1: DNA single-strand base sequences and peptide amino acid sequences involved in the test (X is 5mC)

[0042]

[0043] (2) Prepare standard solutions. SAM and SAH standards (Aladdin Biochemical Technology Co., Ltd., Shanghai, China) with concentration gradients of 0, 0.01, 0.1, 1, 10, 50, and 100 μM were dissolved in 50 mM Tris-HCl, 10 mM NaCl, and 5% glycerol and added to 60 μL with ultrapure water.

[0044] (3) Methyl transfer reaction and termination: The reaction system and standard solution were incubated at 37°C for 90 min, and then repeatedly frozen and thawed in liquid nitrogen for 5 times. 8 μL of proteinase K was added and heated at 55°C for 10 min to completely stop the reaction.

[0045] (4) UPLC-qTOF-MS / MS detection: The reaction solution and standard solution were carefully transferred to a micro-volume plastic liner loaded in a liquid phase injection bottle, and the sample was automatically injected by an autosampler. The detection was performed using Acquity's UPLC-qTOF-MS / MS equipment (Waters Corporation, Milford, USA).

[0046] The chromatographic conditions were as follows: the injection volume of the reaction solution was 5 μL; a CSH C18 column (Acquity UPLC CSHC18 Column, 130 Å, 1.7 µm, 2.1 mm × 100 mm, Waters Corporation, Milford, USA) was used; the mobile phase A was 10 mM ammonium acetate and 0.2% acetic acid, and the mobile phase B was methanol; and a gradient elution was performed for 8 min (total flow rate was 0.3 mL / min, 1 min, 2% B; 5 min, 75% B; 6 min, 2% B).

[0047] The mass spectrometry conditions were as follows: UPLC-qTOF-MS / MS used the positive ion MRM mode to preset the mass-to-charge ratio information of SAM (Precursor ion = 399.15, Fragment ion = 250.1) and SAH (Precursor ion = 385.13, Fragment ion = 136.06); the curtain gas was 35 psi, the ion source gas 1 and gas 2 were both 55 psi, the ion source temperature was 600°C, and the ion spray voltage was 5500 V.

[0048] Finally, the [M+H]+ primary mass spectrum and the corresponding secondary mass spectrum ( Figure 1 ), where the peak area is used for accurate quantification of SAM and SAH.

[0049] (5) Accurate quantification of SAM and SAH: The concentrations of SAM and SAH after the reaction were calculated based on the standard curve obtained from the standard solution data. The results of SAM and SAH in each group were obtained in μM, and a significance analysis was performed based on the t-test ( Figure 2 ).

[0050] (6) Calculation of FaCMT3 activity: In (5), the quantitative results of the concentrations of the SAM and SAH experimental groups were compared with those of the control group, and significant differences were found ( p <0.05) experimental group, the enzyme activity was accurately calculated according to the definition of SAM reduction or SAH increase caused by CMT3 activity per unit time. CMT3 activity was quantitatively calculated according to the following formula:

[0051]

[0052] in, U SAM The activity definition is based on the reduction of SAM content, which indicates the reduction of reactant SAM per unit time due to CMT3 activity, with the unit of pmol SAM / h; U SAH represents the activity definition based on the increase in SAH content, indicating the increase in product SAH per unit time due to CMT3 activity, with the unit of pmol SAH / h; further, c 对照 is the concentration of SAM or SAH in the control group (μM); c 实验 is the concentration of SAM or SAH in the experimental group (μM); v is the total volume of the reaction system (μL); t is the reaction time.

[0053] In this example, UPLC-qTOF-MS / MS successfully and accurately captured the slight changes in SAM and SAH concentrations. The results showed that FaCMT3 only showed in vitro methyl transfer activity on double-stranded DNA substrates rich in CHG and hmCHG sequences. Among them, MS detection showed that after the reaction of the T / A substrate control group, the SAM response intensity peak exceeded 4900 at 1.060 min, while SAH was below the detection limit, indicating that the reaction did not produce SAH; after the reaction of the hmCHG substrate experimental group, the SAM response intensity peak decreased to about 1300, and the SAH response intensity peak increased significantly to about 1800 at 3.407 min ( Figure 1 ), which directly reflects the decrease of reactant SAM and the increase of product SAH caused by the methyl transfer activity of FaCMT3. This method successfully obtained the accurate in vitro activity value of FaCMT3 under the conditions of this example (Table 2), and for the first time obtained the typically defined enzyme activity quantitative calculation value in the activity detection of methyltransferases such as CMT3 with SAM as methyl donor. Among them, since SAH is a reaction product generated from scratch, it can better reflect the enzymatic activity. Therefore, USAH is more recommended as the accurate in vitro activity value of FaCMT3 for reference in the present invention.

[0054] Table 2: Example 1 Figure 2 Results of FaCMT3 methyltransferase activity

[0055]

[0056] Example 2

[0057] This example uses the method disclosed in the present invention to detect the in vitro methyl transfer activity of FaCMT3 and to explore whether H3K9me2 is necessary for FaCMT3 activity. The implementation steps are as follows:

[0058] (1) Construct the reaction system: add 2.5 μM FaCMT3 purified protein, 5 μM double-stranded DNA substrate, 5 μM histone peptide (some groups do not have it), 3 μM SAM, 50 mM Tris-HCl, 10 mM NaCl, 5% glycerol, and ultrapure water to 55 μL. The single-stranded DNA base sequence and peptide amino acid sequence are the same as those in Example 1.

[0059] (2) Prepare standard solutions. Prepare SAM and SAH standards (Sigma-Aldrich, City of Saint Louis, USA) with concentration gradients of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 25, and 50 μM. Dissolve them in 50 mM Tris-HCl, 10 mM NaCl, and 5% glycerol and add ultrapure water to 55 μL.

[0060] (3) Methyl transfer reaction and termination: The reaction system and standard solution were incubated at 37°C for 90 min, and then repeatedly frozen and thawed in liquid nitrogen for three times. 8 μL of proteinase K was added and heated at 55°C for 8 min to completely stop the reaction.

[0061] Steps (4) to (6) are the same as those in Example 1.

[0062] In this example, UPLC-qTOF-MS / MS also successfully and accurately captured the slight changes in SAM and SAH concentrations. The results still showed that FaCMT3 exhibited strong in vitro methyl transfer activity on double-stranded DNA substrates rich in CHG and hmCHG sequences. Among them, the loss of H3K9me2 histone peptides significantly limited the in vitro activity of FaCMT3, but did not completely block the in vitro activity of FaCMT3 (Table 3). In the absence of H3K9me2 histone peptides, FaCMT3 in the hmCHG substrate experimental group still had an in vitro activity of 31.24±3.57pmol SAM / h or 12.53±1.21pmol SAH / h. The results of this embodiment show that the presence of H3K9me2 histone peptide is not a necessary condition for the activity of CMT3 protein in all species. Therefore, the use of histone peptide deficiency as a control group condition may not be universal; this proves the fifth advantage in the content of the invention, that is, compared with the existing method of not adding CMT3 purified protein and not adding H3K9me2 peptide as the control group, the method of the present invention can have a better control effect and serve as a more appropriate background value, thereby obtaining more accurate CMT3 enzyme activity quantitative results, and has the advantages of accuracy and reliable results.

[0063] Table 3: Results of FaCMT3 methyl transfer activity in Example 2

[0064]

[0065] Example 3

[0066] This embodiment adopts the method disclosed in the present invention to detect ( Solanum lycopersicum ) The in vitro methyl transfer activity of purified CMT3 protein (SlCMT3) and the preference of SlCMT3 for different types of DNA substrates were investigated as follows:

[0067] (1) Construct a reaction system: add 1.5 μM SlCMT3 purified protein, 2.5 μM double-stranded DNA substrate, 2.5 μM histone peptide, 3 μM SAM, 50 mM Tris-HCl, 10 mM NaCl, 5% glycerol, and ultrapure water to 60 μL. The DNA single-stranded base sequence and peptide amino acid sequence are the same as those in Example 1.

[0068] (2) Prepare standard solutions. Prepare SAM and SAH standards (Sigma-Aldrich, City of Saint Louis, USA) with a concentration gradient of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 μM. Dissolve them in 50 mM Tris-HCl, 10 mM NaCl, and 5% glycerol, and add ultrapure water to 60 μL.

[0069] (3) Methyl transfer reaction and termination: After the reaction system and standard solution were incubated at 37°C for 60 min, they were repeatedly frozen and thawed in liquid nitrogen for 5 times, 6 μL of proteinase K was added, and the reaction was heated at 55°C for 12 min to completely stop the reaction.

[0070] Steps (4) to (6) are the same as those in Example 1.

[0071] In this example, UPLC-qTOF-MS / MS also successfully and accurately captured the slight changes in SAM and SAH concentrations. The results showed that SlCMT3 showed strong in vitro methyl transfer activity on double-stranded DNA substrates rich in CHG and hmCHG sequences, and also showed weak activity on double-stranded DNA substrates rich in CHH and hmCHH sequences (Table 4). More importantly, this example proves that this method can determine the in vitro activity of CMT3 proteins in different species and has universal applicability.

[0072] Table 4: Results of S1CMT3 methyl transfer activity in Example 3

[0073]

[0074] Example 4

[0075] This embodiment adopts the method disclosed in the present invention to detect tobacco ( Nicotiana benthamiana ) The in vitro methyl transfer activity of purified CMT3 protein (NbCMT3) and the preference of SlCMT3 for different types of DNA substrates were investigated as follows:

[0076] (1) Construct a reaction system: add 3 μM SlCMT3 purified protein, 3.5 μM double-stranded DNA substrate, 3.5 μM histone peptide, 3 μM SAM, 50 mM Tris-HCl, 10 mM NaCl, 5% glycerol, and ultrapure water to 50 μL. The single-stranded DNA base sequence and the peptide amino acid sequence are the same as those in Example 1.

[0077] (2) Prepare standard solutions. Prepare SAM and SAH standards (Aladdin Biochemical Technology Co., Ltd., Shanghai, China) with concentration gradients of 0, 0.1, 0.5, 1, 5, 10, 50, and 100 μM. Dissolve them in 50 mM Tris-HCl, 10 mM NaCl, and 5% glycerol and add ultrapure water to 50 μL.

[0078] (3) Methyl transfer reaction and termination: The reaction system and standard solution were incubated at 37°C for 80 min, and then repeatedly frozen and thawed in liquid nitrogen for three times. 10 μL of proteinase K was added and heated at 55°C for 10 min to completely stop the reaction.

[0079] Steps (4) to (6) are the same as those in Example 1.

[0080] In this example, UPLC-qTOF-MS / MS also successfully and accurately captured the slight changes in SAM and SAH concentrations. The results showed that NbCMT3 protein only showed strong in vitro methyl transfer activity on double-stranded DNA substrates rich in CHG and hmCHG sequences (Table 5). More importantly, this example further demonstrated that this method can determine the in vitro activity of CMT3 proteins from different species and has universal applicability.

[0081] Table 5: Results of NbCMT3 methyl transfer activity in Example 4

[0082]

[0083] The embodiments described above have described in detail the technical solutions and beneficial effects of the present invention. The above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An in vitro detection method for CMT3 protein activity based on accurate quantification of SAM and SAH, characterized in that: The following steps are involved: 1) An in vitro methyl transfer reaction system containing purified CMT3 protein, double-stranded DNA substrate, H3K9me2 histone peptide and S-adenosyl-L-methionine was constructed. The double-stranded DNA substrate contained only TA bases in the control group and CHG bases in the experimental group, H=A / T / G. 2) The reaction system constructed in step 1) was subjected to CMT3 methyl transfer in vitro reaction at 37°C; 3) Using UPLC-qTOF-MS / MS method to accurately quantify SAM, SAH and SAM and SAH standards with different concentration gradients in the reaction system of step 2); The chromatographic conditions were as follows: the injection volume of the reaction system was 5 μL; a CSHC18 column was used; mobile phase A was 10 mM ammonium acetate and 0.2% acetic acid, and mobile phase B was methanol; a gradient elution was performed for 8 min, with a total flow rate of 0.3 mL / min, 1 min, 2% B; 5 min, 75% B; 6 min, 2% B; The mass spectrometry conditions were as follows: UPLC-qTOF-MS / MS used positive ion MRM mode with preset mass-to-charge ratio information of SAM, Precursor ion=399.15, Fragment ion=250.1 and SAH, Precursor ion=385.13, Fragment ion=136.06; curtain gas 35 psi, ion source gas 1 and gas 2 were both 55 psi, ion source temperature 600°C, ion spray voltage 5500 V; 4) According to the quantitative results of step 3), the in vitro methyl transfer activity was calculated. Specifically, the quantitative results of the SAM and SAH experimental groups were compared with the results of the control group, and combined with the significance analysis, it was determined whether there was a significant difference with the control group (p < 0.05). If there was a significant difference, the CMT3 activity was quantitatively calculated according to the following formula: ； Among them, USAM is the activity definition based on the reduction of SAM content, which means the reduction of reactant SAM per unit time due to CMT3 activity, and the unit is pmol SAM / h; USAH is the activity definition based on the increase of SAH content, which means the increase of product SAH per unit time due to CMT3 activity, and the unit is pmol SAH / h; ccontrol is the concentration of SAM or SAH in the control group μM; cexperiment is the concentration of SAM or SAH in the experimental group μM; v is the total volume of the reaction system μL; t is the reaction time.

2. The in vitro detection method for CMT3 protein activity based on accurate quantification of SAM and SAH according to claim 1, characterized in that In step 1), the amount of CMT3 purified protein added to the in vitro methyl transfer reaction system is 1 to 5 μM, the amount of double-stranded DNA substrate added is 1 to 5 μM; the amount of H3K9me2 histone peptide added is 1 to 5 μM, and the amount of S-adenosyl-L-methionine added is 1 to 3 μM.

3. The in vitro detection method for CMT3 protein activity based on accurate quantification of SAM and SAH according to claim 1, characterized in that In step 1), the in vitro methyl transfer reaction system also contains 5-20 mM sodium chloride, 3-6% glycerol, and ultrapure water is added to a total volume of 40-60 μL. The pH value of the system is 7.5-8.5, and the mixture is gently stirred to mix.

4. The in vitro detection method for CMT3 protein activity based on accurate quantification of SAM and SAH according to claim 1, characterized in that In step 2), incubate at 37°C for 30-120 min, then freeze-thaw repeatedly in liquid nitrogen 2-5 times, add 5-10 μL proteinase K, and heat at 55°C for 5-15 min to completely stop the reaction.

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