CN115825214A - MALDI-TOF-MS-based method for in-vitro screening of hMAT2A protein inhibitor and application thereof - Google Patents
MALDI-TOF-MS-based method for in-vitro screening of hMAT2A protein inhibitor and application thereof Download PDFInfo
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Abstract
The invention provides a MALDI-TOF-MS-based method for in vitro screening of hMAT2A protein inhibitor and application thereof, relating to the technical field of stable isotope labeled new drug discovery. The invention provides a method for screening hMAT2A protein inhibitor in vitro based on MALDI-TOF-MS, which directly measures the signal ratio of an enzyme catalysis product SAM and SAM-D3 of an isotope label through MALDI-TOF-MS to relatively quantify and qualify the effect of the inhibitor, overcomes the false positive result caused by ATP easy hydrolysis by detecting the ATP content of a reactant, solves the system error by introducing the isotope label, has simple operation and short detection time (1 minute/sample); the sample target of MALDI-TOF-MS can be recycled, thereby realizing high-throughput screening of MAT2A inhibitor.
Description
Technical Field
The invention relates to the technical field of stable isotope labeling new drug discovery, in particular to a method for in vitro screening of hMAT2A protein inhibitor based on MALDI-TOF-MS and application thereof.
Background
Methionine adenosyltransferase 2A (Methionine adenosyltransferase 2a, mat 2a) is a rate-limiting enzyme in the Methionine cycle, and primarily catalyzes the synthesis of S-adenosylmethionine (SAM) from Methionine and Adenosine Triphosphate (ATP). Human MAT exists in the cell as a heterotetramer, consisting of a homodimer of hMAT2A and two regulatory subunits (hMAT 2 β) that form the catalytic unit. MAT2A forms a functional homodimer in its purified, active form and binds to the regulatory protein MAT 2B. MAT2B increased the sensitivity of MAT2A to inhibition by SAM products to modulate the activity of MAT2A, but did not significantly enhance its activity. MAT2A is ubiquitously expressed in human cell types and is the predominant form in human tumors. According to cell localization studies MAT2A is present both in the cytoplasm and in the nucleus. Nuclear enrichment with MAT2A occurs during replication and subsequent G2 phase, meeting the requirement for high methylation of DNA and histone methylation processes in the S phase nuclei.
SAM is the major methyl donor for the synthesis of polyamines and glutathione in nucleic acids, phospholipids, histones, biogenic amines, and protein methylation, an important modification of proteins and nucleic acids, which controls DNA transcription (via direct DNA methylation and indirectly via histone methylation), RNA stability and localization, and protein activity, structure, localization, and protein/protein interactions. Endogenous metabolites and exogenous drugs are also methylated, changing their physicochemical properties and biological activities. Methylation is closely related to many diseases such as cancer, aging, senile dementia, etc. Regulation of SAM biosynthesis can affect cell growth, differentiation and function. At present, the activity of SAM in the transmethylation reaction is confirmed as a rate-limiting factor of the development of lung cancer stem cells, which also makes MAT2A and related enzymes of methionine cycle become targets of anticancer drugs.
MAT2A protein is increased in expression in cancers such as colon cancer, liver cancer, stomach cancer, blood and liver. About 15% of human cancers show a deletion of the MTAP (S-methyl-5' -thioadenosine phosphorylase) gene, and thus lack a methionine recovery pathway for polyamine synthesis. Deletion of MTAP in chr9p21 typically includes deletion of the CDKN2a oncosuppressor locus. Combined genetic lethality of MATP-/-cancer cells, studies have shown that these cancer cells have increased sensitivity to inhibition by MAT2A, PRMT and PRMT 1. This finding led to the search for effective drugs against these enzymes, and at present, 2 drugs against MAT2A have been put into clinical trials, one against lymphoma and solid tumor AG-2703 and one against solid tumor IDE-397, which provide hopes and motivation for MAT2A as a target for the treatment of various cancers, but also indicate that MAT2A inhibitors remain a gap, and therefore, the search for a high-throughput screening method for MAT2A inhibitors is of great importance.
At present, the method for in vitro screening MAT2A inhibitor mainly comprises the steps of detecting enzymatic chemical reaction of SAM, and indirectly measuring phosphate generated by the reaction by using a phosphate measurement kit, so as to evaluate the MAT2A inhibition activity of the drug. For example, in the phosphate content measurement, the absolute product amount is measured by comparing with a standard curve of potassium phosphate buffer (pH 8.028), and this method has limitations such as excessive cost, long time, complicated operation steps, and the like.
At present, a stable isotope chemical labeling method has been widely applied to high-throughput Mass Spectrometry (MS) -based quantitative proteomics for biological and clinical applications, and has the advantages of elimination of system errors, safety, no radiation, quantitative analysis and the like. Meanwhile, matrix-assisted laser-induced desorption/Ionization Mass Spectrometry (MALDI-TOF-MS) is a high-sensitivity soft Ionization Mass Spectrometry technology for biomolecule analysis, and the MALDI-TOF-MS is the most advantageous in simplicity, rapidness, accuracy and low cost and plays a key role in analysis of biomacromolecules including peptides, proteins, nucleic acids, small molecules and the like. MALDI-TOF-MS has the characteristics of high resolution, high flux, high sensitivity, high salt tolerance and small sample volume, and provides powerful technical support for high-flux screening of drugs.
Disclosure of Invention
In view of this, the present invention aims to provide a method for in vitro screening of an hMAT2A protein inhibitor based on MALDI-TOF-MS, which can rapidly obtain the effect of a drug to be detected, thereby rapidly determining the inhibition effect of the drug to be detected on the hMAT2A protein.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a MALDI-TOF-MS-based method for in vitro screening of an hMAT2A protein inhibitor, which comprises the following steps:
mixing and incubating a drug to be tested, hMAT2A and a reaction solution, adding ATP and L-Met for reaction, and centrifuging to obtain an experimental group supernatant;
mixing and incubating secondary purified water, hMAT2A and reaction solution, and adding ATP and L-Met-D 3 Reacting, and centrifuging to obtain a control group supernatant;
and mixing the experimental group supernatant and the control group supernatant to obtain a sample to be tested, mixing the sample to be tested with the matrix, performing MALDI-TOF-MS test, and comparing the peak intensities of the experimental group and the control group to judge the inhibition effect of the drug to be tested on hMAT2A protein.
Preferably, the reaction solution consists of 100mM Hepes solution, 50mM KCl solution and 10mM MgCl 2 And (4) solution composition.
Preferably, the incubation temperature is 20-30 ℃ and the incubation time is 30-60min.
Preferably, the volume ratio of the drug to be detected, the hMAT2A and the reaction solution is (0.5-1): (0.2-1): 6.2-7.5); the volume ratio of the drug to be detected, ATP and L-Met is (0.5-1) to 1 (0.8-1).
Preferably, the reaction temperature is 20-30 ℃ and the reaction time is 30-60min.
Preferably, the volume ratio of the experimental group supernatant to the control group supernatant is 1:1.
Preferably, the volume ratio of the sample to be tested to the matrix is 1:1.
Preferably, the matrix is any one of THAP, DHB or CHCA.
Preferably, the conditions of the MALDI-TOF-MS test are as follows: the molecular weight scanning range is 0-1000 kDa, and the laser energy is 12-36%.
The invention also provides application of the method in-vitro high-throughput screening of the hMAT2A protein inhibitor.
The invention has the beneficial effects that:
the invention provides a method for in vitro screening of hMAT2A protein inhibitor based on MALDI-TOF-MS, wherein the signal ratio of an enzyme catalysis product SAM and SAM-D3 marked by an isotope is directly measured by MALDI-TOF-MS to relatively quantify and qualify the effect of the inhibitor, the direct detection of the product overcomes the false positive result caused by the easy hydrolysis of ATP due to the detection of the ATP content of a reactant, and the introduction of the isotope mark solves the system error; meanwhile, MALDI-TOF-MS is simple to operate, and the detection time is short (1 minute/sample); the sample target of MALDI-TOF-MS can be recycled, thereby realizing high-throughput screening of MAT2A inhibitor. Experiments prove that the method can quickly obtain the effect of the drug to be detected in each group of experiments, so that the inhibition effect of the drug on the hMAT2A protein can be quickly judged, and the method has the advantages of simplicity in operation, low enzyme consumption, high accuracy and good repeatability.
Drawings
FIG. 1 is a chemical structural diagram of AG-270 and Tannic acid.
FIG. 2 is a diagram showing the mode of action of in vitro biosynthesis isotope labeled SAM of experimental group and drug-adding group.
FIG. 3 is a Western blot qualitative result chart of hMAT 2A.
FIG. 4 is a graph showing the peak area changes of a group of isotopically labeled SAM generated by the action of AG-270, a positive drug, on hMAT2A at different concentrations.
FIG. 5 is a graph of IC50 values for AG-270 versus hMAT2A simulated using the method of the present invention.
FIG. 6 is a diagram showing the screening results of medicines No. 1-10 among ten natural product mixed medicines E-E.
FIG. 7 shows the IC simulated by the method of the present invention for No.1 drug Tannic acid in E-E selected by screening 50 Graph is shown.
Detailed Description
The invention provides a MALDI-TOF-MS-based method for in vitro screening of an hMAT2A protein inhibitor, which comprises the following steps:
mixing and incubating a drug to be tested, hMAT2A and a reaction solution, adding ATP and L-Met to react, and centrifuging to obtain an experimental group supernatant;
mixing and incubating secondary purified water, hMAT2A and reaction solution, and adding ATP and L-Met-D 3 Reacting, and centrifuging to obtain a control group supernatant;
mixing the experimental group supernatant and the control group supernatant to obtain a sample to be tested, mixing the sample to be tested with the matrix, performing MALDI-TOF-MS test, and comparing the peak intensities of the experimental group and the control group to judge the inhibition effect of the drug to be tested on hMAT2A protein.
In the present invention, the reaction solution is preferably composed of 100mM Hepes solution, 50mM KCl solution and 10mM MgCl 2 Solution composition; the pH of the Hepes solution is preferably 8.5. In the present invention, the incubation temperature is preferably 20 to 30 ℃, more preferably 25 ℃, and the time is preferably 30 to 60min, more preferably 40 to 50min. In the invention, the volume ratio of the drug to be detected, the hMAT2A and the reaction solution is preferably (0.5-1): (0.2-1): 6.2-7.5, more preferably 1.5; the volume ratio of the drug to be tested, ATP and L-Met is preferably (0.5-1): 1, (0.8-1), more preferably 1. In the invention, the hMAT2A is preferably hMAT2A protein after expression and purification, and the concentration of the hMAT2A protein is preferably 2 mug/muL; the concentration of ATP is preferably 5mM; the L-Met and L-Met-D 3 The concentration of (labeled with deuterium atom) is preferably 5mM.
In the present invention, the reaction temperature is preferably 20 to 30 ℃, more preferably 25 ℃, and the reaction time is preferably 30 to 60min, more preferably 60min. In the present invention, the quenching reaction is preferably performed after the reaction, and the solvent for the quenching reaction is preferably acetonitrile. In the present invention, the centrifugation is preferably performed at 4 ℃ for 5min.
The invention mixes the experimental group supernatant and the control group supernatant to obtain a sample to be tested, and the sample to be tested is mixed with the matrix to carry out MALDI-TOF-MS test. In the invention, the volume ratio of the experimental group supernatant to the control group supernatant is preferably 1:1; the volume ratio of the sample to be tested to the matrix is preferably 1:1. In the present invention, the matrix is preferably any one of THAP, DHB or CHCA. In the present invention, the MALDI-TOF-MS mode is preferably a linear mode and a positive charge mode suitable for biological small molecules (amino acids, nucleotides, monosaccharides); the conditions of the MALDI-TOF-MS are preferably as follows: the molecular weight scanning range is 0-1000 kDa, and the laser energy is 12-36%.
The invention also provides application of the method in-vitro high-throughput screening of the hMAT2A protein inhibitor.
The method for in vitro screening the hMAT2A protein inhibitor based on MALDI-TOF-MS utilizes a stable isotope labeling method to biosynthesize SAM and SAM-D in vitro 3 (deuterium atom labeling), and then detecting SAM and SAM-D by mass spectrometry 3 The ratio of the peak intensities of the two signals reflects the inhibition effect of the drug to be detected on the hMAT 2A.
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
In the following examples, unless otherwise specified, all methods are conventional.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Preparation of MAT2A protein
Human MAT2A protein with high tolerance at 37 ℃ is selected for experiment, and prokaryotic expression protein technology with high expression level is selected for expressing the protein.
1. Cloning the self-constructed full-length hMAT2A-2Strep sequence into a pET22b (+) vector with a C-terminal 6 (His) tag to construct a pET22b (+) -hMAT2A-2Strep plasmid; wherein the sequence of the hMAT2A-2Strep is shown as follows: ATGAACGGACAGCTCAACGGCTTCCACGAGGCGTTCATCGAGGAGGGCACATTCCTTTTCACCTCAGAGTCGGTCGGGGAAGGCCACCCAGATAAGATTTGTGACCAAATCAGTGATGCTGTCCTTGATGCCCACCTTCAGCAGGATCCTGATGCCAAAGTAGCTTGTGAAACTGTTGCTAAAACTGGAATGATCCTTCTTGCTGGGGAAATTACATCCAGAGCTGCTGTTGACTACCAGAAAGTGGTTCGTGAAGCTGTTAAACACATTGGATATGATGATTCTTCCAAAGGTTTTGACTACAAGACTTGTAACGTGCTGGTAGCCTTGGAGCAACAGTCACCAGATATTGCTCAAGGTGTTCATCTTGACAGAAATGAAGAAGACATTGGTGCTGGAGACCAGGGCTTAATGTTTGGCTATGCCACTGATGAAACTGAGGAGTGTATGCCTTTAACCATTGTCTTGGCACACAAGCTAAATGCCAAACTGGCAGAACTACGCCGTAATGGCACTTTGCCTTGGTTACGCCCTGATTCTAAAACTCAAGTTACTGTGCAGTATATGCAGGATCGAGGTGCTGTGCTTCCCATCAGAGTCCACACAATTGTTATATCTGTTCAGCATGATGAAGAGGTTTGTCTTGATGAAATGAGGGATGCCCTAAAGGAGAAAGTCATCAAAGCAGTTGTGCCTGCGAAATACCTTGATGAGGATACAATCTACCACCTACAGCCAAGTGGCAGATTTGTTATTGGTGGGCCTCAGGGTGATGCTGGTTTGACTGGACGCAAAATCATTGTGGACACTTATGGCGGTTGGGGTGCTCATGGAGGAGGTGCCTTTTCAGGAAAGGATTATACCAAGGTCGACCGTTCAGCTGCTTATGCTGCTCGTTGGGTGGCAAAATCCCTTGTTAAAGGAGGTCTGTGCCGGAGGGTTCTTGTTCAGGTCTCTTATGCTATTGGAGTTTCTCATCCATTATCTATCTCCATTTTCCATTATGGTACCTCTCAGAAGAGTGAGAGAGAGCTATTAGAGATTGTGAAGAAGAATTTCGATCTCCGCCCTGGGGTCATTGTCAGGGATCTGGATCTGAAGAAGCCAATTTATCAGAGGACTGCAGCCTATGGCCACTTTGGTAGGGACAGCTTCCCATGGGAAGTGCCCAAAAAGCTTAAATATGCGGCCGCATGGAGCCACCCGCAGTTCGAGAAAGGTGGAGGTTCGGGAGGTGGATCGGGTGGAAGCGCTTGGAGCCACCCGCAGTTCGAGAAA (SEQ ID NO. 1).
2. The plasmid is expressed in BL21 Escherichia coli strain
2.1 conversion: adding 0.5 μ L plasmid into BL21 strain, incubating for 30min on ice, heat-shocking at 42 deg.C for 45-60s, standing on ice for 2-3min, adding 200 μ L culture medium, coating plate, and incubating overnight in 37 deg.C constant temperature incubator;
2.2 shaking for a short time: selecting a point growing to be round on the plate, gently picking the point by using a gun head, beating the gun head into 20ml of 2YT culture medium, adding an ampicillin antibody (1000;
2.3 amplification: the method comprises the following steps of 1:100 ml of the shake culture medium was added to 1L 2YT medium, to which ampicillin antibody was added. Shaking at 37 deg.C for 4h until OD value of bacterial liquid reaches 0.5, adding IPTG to final concentration of 0.5mM, and shaking at 37 deg.C for 2h;
2.4, collecting bacteria: centrifuging the bacterial solution at 8000g,4 deg.C for 10min, removing the supernatant, adding 30ml of lysate (100 mM Tris/HClpH7.5, 150mM NaCl,1mM EDTA), placing in a refrigerator at-80 deg.C, and freeze thawing for three times;
2.5 ultrasonic disruption of bacterial liquid: performing ultrasonic treatment with contact probe at 40% power for 10s, stopping for 20s, repeating for 12 times, and centrifuging at 14000g and 4 deg.C for 20min; collecting supernatant in the tube to a new centrifuge tube
Adding beads into a centrifuge tube, and turning over and incubating overnight at 4 ℃;
2.6 impurity washing: centrifuging the incubated solution at 1000g,4 ℃ for 1min, removing a little supernatant, transferring the rest incubation system to a column, adding 6mL of Wash buffer (100 mM Tris/HCl pH7.5, 500mM NaCl,1mM EDTA) after the lysate naturally drips, repeatedly washing for 6 times after the lysate naturally drips, and washing off the hybrid proteins bound on the beads;
2.7 elution: connecting a clean centrifuge tube below the column, adding 600 μ L of Elute buffer (100 mM Tris/HCl pH7.5, 150mM NaCl,1mM EDTA,2.5mM desthiobatin) into the column, taking away the plug after the Elute buffer is fully combined with beads for 9-10min, allowing the eluate to naturally flow through and drip into the collection tube below, and repeating elution twice;
3. the protein concentration is measured by a NanoDrop One/OneC trace nucleic acid protein concentration measuring instrument and then split charging is carried out for storage at the temperature of minus 80 ℃.
Example 2
In vitro biosynthesis experimental method of SAM catalyzed by MAT2A
In vitro biosynthesis experiment reaction liquid RB matched with SAM catalyzed by MAT 2A: containing 100mM hepes, pH 8.5, 50mM KCl,10mM MgCl 2 。
MAT2A catalyzed SAM in vitro biosynthesis experiments, according to the table 1 preparation of 10 u L reaction system.
TABLE 1 reaction System
Mixing a drug to be detected, RB and hMAT2A, incubating for 60min at normal temperature, adding L-Met and ATP to start reaction, reacting for 60min at normal temperature, adding acetonitrile with the same volume to quench reaction, and centrifuging for 5min at 1200rpm and 4 ℃ to obtain an experimental group supernatant;
mixing the secondary purified water, RB and hMAT2A, incubating for 60min at normal temperature, and adding L-Met-D 3 Starting reaction with ATP, reacting at normal temperature for 60min, adding equal volume of acetonitrile, quenching reaction, centrifuging at 1200rpm at 4 deg.C for 5min to obtain control group supernatant;
and mixing the experimental group supernatant and the control group supernatant according to the volume ratio of 1:1 to obtain a sample to be tested.
MALDI-TOF-MS detection analysis
And mixing 6 mu L of samples to be tested with 6 mu L of matrix (THAP, DHB, CHCA and 5 mg/mL), spotting, completely drying, testing, and performing mass spectrometry to determine whether SAM is generated. The MALDI-TOF-MS mode selects a linear mode and a positive charge mode, the molecular weight scanning range is set to be 0-1000 kDa, and if the signal detection is weak, the detection gain (controlled within 2 times) can be properly improved, and the energy of the laser is 12-36%.
3. Result processing
The signal value of each peak in the mass spectrum obtained by MALDI-TOF-MS, SAM-D can be directly obtained by mass spectrum processing software 3 The difference of the relative molecular mass of SAM is about 3kDa, and SAM-D is corresponding to the sample without drug treatment 3 The signal value of (c), the sample treated with the kit corresponds to the signal value of SAM. The signal ratio of the drug-added group to the drug-not-added group was used as the evaluation of the drug inhibition effect. Lower values indicate a stronger inhibitory effect of the drug on MAT2A, and lower SAM formation. At the same time, SAM and SAM-D are treated according to different drug concentrations 3 The signal ratio of (A) was plotted to calculate the IC of the drug 50 。
Example 3
Taking AG-270 entering clinical trial as an example
FIG. 2 is a diagram of the action pattern of SAM biosynthesis in vitro between the drug-added group and the drug-free group, and the results show that: the core of the experiment lies in that the hMAT2A protein extracted by escherichia coli can be used for successfully catalyzing and synthesizing SAM and SAM-D3, the signal values of the SAM and the SAM-D3 can be detected by mass spectrometry under the condition of not being interfered by a matrix peak, and the ratio of the SAM and the SAM-D3 reflects the inhibition effect of the drug to be detected. The inhibition effect of the drug to be screened on hMAT2A can be rapidly judged by utilizing the characteristics that MALDI-TOF-MS can be used for automatic and high-throughput detection. In this example, MAT2A inhibitor AG-270 was selected as a positive drug for the study.
The experiment was divided into two steps, the first step using plasmid pET22b (+) -hMAT2A-2Strep to express purified hMAT2A protein, the specific steps are as described in example 1. FIG. 3 is a diagram of an immunoblot experiment for purification of extracted hMAT2A, showing that: the purified protein was confirmed to be hMAT2A, and the protein was quantified at a level of about 3.1 μ g/μ L.
The second step is an in vitro biosynthesis experiment of SAM, and the reaction system is shown in Table 2:
TABLE 2SAM in vitro biosynthesis experiment reaction System
According to the steps of example 2, AG-270, RB and hMAT2A protein are mixed and incubated for 60min at normal temperature (25 ℃), L-Met and ATP are added for starting reaction, 10 microliter of acetonitrile is added for quenching reaction after the reaction is carried out for 60min at normal temperature, and centrifugation is carried out for 5min at 1200rpm and 4 ℃ to obtain experimental group supernatant;
mixing the secondary purified water, RB and hMAT2A, incubating for 60min at normal temperature, and adding L-Met-D 3 Starting reaction with ATP, reacting at normal temperature for 60min, adding 10 μ L acetonitrile, quenching reaction, centrifuging at 1200rpm at 4 deg.C for 5min to obtain control group supernatant;
3 mu.L of each of the control group supernatant and the experimental group supernatant was mixed with 6 mu.L of CHCA (2 mg/ml) to obtain a sample to be tested.
The third step is MALDI-TOF-MS detection and result processing, the peak area change of the obtained result positive drug AG-270 acting on hMAT2A with different concentrations is shown in figure 4, the peak of SAM is at 400.171Da, and the isotopically labeled SAM-D is at 403.205Da 3 When the positive drug AG-270 acts on the hMAT2A at different concentrations, the signal ratio of the two peaks changes, which reflects the change of the activity of the hMAT2A at different drug concentrations. IC was calculated as AG-270 as shown in FIG. 5 50 The value is 534.7nM, demonstrating the feasibility of the method of the invention.
Example 4
The method of the invention is used to screen the inhibition effect of part of natural product libraries on hMAT2A protein by using AG-270 in example 3 as a positive drug, and finally, drugs with inhibition activity are screened from E in ten mixed drugs, as shown in FIG. 6. The specific experimental procedure was similar to that of example 3, and the drug Tannic Acid (TA) having inhibitory activity against hMAT2A was selected.
The reaction system of SAM in vitro biosynthesis experiment is shown in the following table 3:
TABLE 3 reaction System
According to the steps of the embodiment 2, tannic acid, reaction solution RB and hMAT2A protein are mixed and incubated for 60min at normal temperature (25 ℃), then L-Met and ATP are added to start reaction, 10 mu L of acetonitrile is added to quench reaction after the reaction is carried out for 60min at normal temperature, centrifugation is carried out for 5min at 1200rpm and 4 ℃, and the supernatant of the experimental group is obtained;
mixing the secondary purified water, RB and hMAT2A, incubating for 60min at normal temperature, and adding L-Met-D 3 Starting reaction with ATP, reacting at normal temperature for 60min, adding 10 μ L acetonitrile, quenching reaction, centrifuging at 1200rpm at 4 deg.C for 5min to obtain control group supernatant;
3 mu.L of each of the control group supernatant and the experimental group supernatant was mixed with 6 mu.L of CHCA (2 mg/ml) to obtain a sample to be tested.
The test samples were tested and the data analyzed according to the method described in example 2. After processing the data, FIG. 7 shows the IC of Tannic acid drugs with inhibitory effect on hMAT2A protein, selected by the method of the present invention 50 Value, IC thereof 50 The value is 4.239 mu M, and when the concentration reaches 50 mu M, the inhibition effect is as high as 80%.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A method for in vitro screening of hMAT2A protein inhibitor based on MALDI-TOF-MS, which comprises the following steps:
mixing and incubating a drug to be tested, hMAT2A and a reaction solution, adding ATP and L-Met for reaction, and centrifuging to obtain an experimental group supernatant;
mixing and incubating secondary purified water, hMAT2A and reaction solution, and adding ATP and L-Met-D 3 Reacting, and centrifuging to obtain a control group supernatant;
mixing the experimental group supernatant and the control group supernatant to obtain a sample to be tested, mixing the sample to be tested with the matrix, performing MALDI-TOF-MS test, and comparing the peak intensities of the experimental group and the control group to judge the inhibition effect of the drug to be tested on hMAT2A protein.
2. The method according to claim 1, wherein the reaction solution is prepared from a 100mM Hepes solution, a 50mM KCl solution and 10mM MgCl 2 And (4) solution composition.
3. The method according to claim 1, wherein the incubation is at a temperature of 20-30 ℃ for a period of 30-60min.
4. The method of claim 1, wherein the volume ratio of the drug to be tested, hMAT2A and the reaction solution is (0.5-1): (0.2-1): 6.2-7.5); the volume ratio of the drug to be detected, ATP and L-Met is (0.5-1) to 1 (0.8-1).
5. The method according to claim 1, wherein the reaction temperature is 20-30 ℃ and the reaction time is 30-60min.
6. The method of claim 1, wherein the ratio of the volume of the experimental group supernatant to the control group supernatant is 1:1.
7. The method of claim 1, wherein the volume ratio of the sample to be tested to the matrix is 1:1.
8. A method according to claim 1, wherein the matrix is any one of THAP, DHB or CHCA.
9. The method according to claim 1, wherein the conditions of the MALDI-TOF-MS test are: the molecular weight scanning range is 0-1000 kDa, and the laser energy is 12-36%.
10. Use of the method of any one of claims 1-9 for high throughput screening of hMAT2A protein inhibitors in vitro.
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