阅读说明：本技术 一种天然产物药物作用靶点的识别方法 (Method for identifying action target of natural product medicine ) 是由 李静 戚欣 买小圆 唐薇 于 2019-09-30 设计创作，主要内容包括：本发明提出了一种天然产物药物作用靶点的识别方法。本发明包括以下步骤：1)取待测药物和对照溶剂分别添加于有效细胞中,共同孵育；2)置于30-80℃下,热处理3-10min,裂解,离心,得上清液；3)电泳,转印；4)采用广谱的蛋白抗体或染料分别染色,并对比,寻找蛋白稳定差异的条带；5)另取待测药物蛋白样电泳,将蛋白稳定差异的条带质谱分析,验证,得靶点。本发明是一种基于蛋白热稳定与广谱分子染色的天然产物药物作用靶点识别方法,可以高效、低成本、免标记地识别潜在的药物靶点,天然产物的用量少,发现潜在的药物靶点不仅可以推动疾病作用机制和药理学研究,还可以为药物潜在的副作用和药品的商业化提供指导信息。(The invention provides a method for identifying a natural product drug action target. The invention comprises the following steps: 1) adding the drug to be detected and the control solvent into the effective cells respectively, and incubating together; 2) placing at 30-80 deg.C, heat treating for 3-10min, cracking, centrifuging to obtain supernatant; 3) electrophoresis and transfer printing; 4) respectively staining by using broad-spectrum protein antibodies or dyes, comparing, and searching for bands with protein stability differences; 5) and carrying out electrophoresis on the drug protein sample to be detected, and carrying out mass spectrometry on the bands with protein stability difference and verifying to obtain a target spot. The invention relates to a natural product drug action target recognition method based on protein thermal stability and broad spectrum molecular dyeing, which can efficiently recognize potential drug targets at low cost in a label-free manner, the dosage of natural products is small, and the discovery of the potential drug targets can not only promote the research of disease action mechanisms and pharmacology, but also provide guidance information for the potential side effects of drugs and the commercialization of drugs.)

1. A method for identifying action targets of natural product drugs is characterized by comprising the following steps: the method comprises the following steps:

1) collecting a sample, and dissolving a drug to be detected in a solvent to obtain an experimental sample; taking another solvent as a control sample; respectively adding the experimental sample and the control sample into the effective cells, and incubating for 1-5h to respectively obtain a drug group sample to be detected and a solvent control group sample;

2) performing heat treatment, namely placing the drug group sample to be detected and the solvent control group sample at 30-80 ℃, performing heat treatment for 3-10min, cracking, centrifuging, and taking supernate to respectively obtain a drug protein sample to be detected and a solvent control protein sample;

3) performing electrophoresis and transfer printing, namely performing electrophoresis and transfer printing on the drug protein sample to be detected and the solvent control protein sample to obtain a drug transfer printing group to be detected and a solvent control transfer printing group;

4) dyeing, namely dyeing the drug transfer printing group to be detected and the solvent control transfer printing group by adopting a broad-spectrum protein antibody or dye, comparing, and searching for bands with protein stability differences;

5) and (3) performing mass spectrometry, namely performing electrophoresis on another drug protein sample to be detected, performing mass spectrometry on the bands with protein stability difference, and verifying to obtain a target point.

2. The method for identifying the natural product drug action target according to claim 1, wherein the step 4) comprises the following steps:

a) preparing 5% solution of skimmed milk powder with 1 × TBST buffer solution;

b) taking a drug transfer group to be tested and a solvent contrast transfer group, adding the solution obtained in the step a), and incubating for 1-2h at room temperature on a shaking table;

c) washing with 1 × TBST buffer solution, adding protein antibody or dye, standing at 4 deg.C, and incubating overnight;

d) coating an ECL luminescent reagent or a dye substrate under the condition of keeping out of the sun, and developing by adopting an imaging system;

e) comparing the transfer printing group of the drug to be tested with the solvent control transfer printing group, searching for a strip with protein stability difference, and recording the molecular weight.

3. The method for identifying natural product drug action targets according to claim 2, wherein the method comprises the following steps:

the protein antibody is any one of a broad-spectrum tyrosine protein kinase antibody, a serine/threonine protein kinase antibody, a protein silver stain and biotin-labeled lectin.

4. The method for identifying natural product drug action targets according to claim 2, wherein the method comprises the following steps:

in step c), when protein antibody is added, the protein antibody is a primary antibody, and the primary antibody further comprises the following operations after overnight incubation:

taking a secondary antibody, and diluting the secondary antibody by 2000-fold with 1 xTBST buffer solution; and (3) washing the transfer printing group of the drug to be detected and the solvent control transfer printing group by adopting a 1 xTBST buffer solution, adding the diluted secondary antibody into the washed transfer printing group of the drug to be detected and the solvent control transfer printing group in a shaking table at room temperature within 1-2h, slowly shaking at room temperature, and incubating for 1-2 h.

5. The method for identifying the natural product drug action target according to any one of claims 1 to 4, wherein the electrophoresis in the step 3) is specifically performed by:

A) taking SDS-PAGE gel, wherein the SDS-PAGE gel comprises 3-5% concentrated gel and 8-12 separating gel, coating and forming pores;

B) adding 8-10 mu L of a drug protein sample to be detected or a solvent control protein sample into each hole, and adding protein pre-staining Marker into the side holes;

C) placing in ice water bath, performing electrophoresis at 60-80V for 20-30min, after the protein pre-staining Marker is completely separated, increasing the voltage to 120-130V, and continuing electrophoresis.

6. The method for identifying the natural product drug action target according to claim 1, wherein the heat treatment in the step 2) comprises the following specific operations:

s1 enriched cells: washing the drug group sample to be detected and the solvent control group sample to respectively obtain a drug group cell to be detected and a solvent control group cell;

s2 gradient heating: respectively heating the cells of the drug group to be detected and the cells of the solvent control group in a gradient manner to 30-80 ℃ to obtain cell suspension of the drug group to be detected and cell suspension of the solvent control group;

s3 cell lysis: cracking the cell suspension of the drug group to be detected and the cell suspension of the solvent control group, centrifuging and collecting supernatant;

s4 sample collection: adding equal volume of Loading Buffer into the supernatant respectively, resuspending, decocting in boiling water for 15-20min, standing at-20 deg.C, and storing.

7. The method for identifying natural product drug action targets according to claim 6, wherein the method comprises the following steps:

the cracking is any one of repeated freeze thawing of liquid nitrogen, cracking of cell lysate and ultrasonic disruption cracking.

8. The method for identifying natural product drug action targets according to claim 6, wherein the method comprises the following steps:

in step S2, during gradient heating, the cells of the drug group to be tested and the cells of the solvent control group are heated to 40-60 ℃ in a gradient manner.

9. The method for identifying the natural product drug action target according to claim 1, wherein in the step 1), the specific operation of sample collection is as follows:

sa cell plating: digesting and counting effective cells in good growth state and logarithmic growth phase with pancreatin, inoculating, and inoculating at 37 deg.C and 5% CO₂Culturing for 24h under the condition;

adding Sb cells: setting the experimental sample and the control sample, respectively, and adding the effective cells cultured in step Sa at 37 deg.C and 5% CO₂Continuously culturing for 1-3h under the condition;

sc-collected cells: removing the culture medium, adding cold PBS, washing, adding pancreatin for digestion, and uniformly blowing to make all adherent cells suspended.

10. The method for identifying the natural product drug action target according to claim 1, wherein the mass spectrometry in the step 5) is specifically performed by:

and (3) taking a drug protein sample to be detected, carrying out electrophoresis according to the step 3), wherein the sample loading amount during electrophoresis is 20-30 mu L, cutting off adhesive strips near the difference, rinsing with ultrapure water, and carrying out mass spectrometry.

Technical Field

The invention relates to the technical field of drug target identification, in particular to a method for identifying a natural product drug action target.

Background

The natural product refers to components or metabolites thereof in animals, plant extracts (plant extracts for short) or insects, marine organisms and microorganisms, and a plurality of endogenous chemical components in human bodies and animals, and mainly comprises proteins, polypeptides, amino acids, nucleic acids, various enzymes, monosaccharides, oligosaccharides, polysaccharides, glycoproteins, resins, colloidal substances, lignin, vitamins, fats, oils, waxes, alkaloids, volatile oils, flavones, glycosides, terpenes, phenylpropanoids, organic acids, phenols, quinones, lactones, steroids, tannins, antibiotics and other naturally-occurring chemical components. Natural products with unique chemical properties and a wide range of biological activities often have novel modes of action that are hard to imagine when designing synthetic libraries, and become an important source of novel drug leads. From 1981 to 2014, 33% of all approved natural product drugs are natural products or their direct semisynthetic derivatives. Due to the great difference of ecological environment, the chemical structure and biological activity of marine natural products are richer than those of terrestrial organisms, and the marine natural products are an important source of marine medicines. By the end of 2012, about 2.5 million marine natural products have been found throughout the world. Since 2007, new marine natural products are published in average quantities over 1000 years, and 50% of these marine natural products have been found to have various biological activities. More encouraging, marine natural products with an ever-new framework have been reported.

The early definition of the action target of the new drug or the active compound is beneficial to carrying out optimization and reconstruction aiming at the target, establishing a proper drug evaluation model and searching corresponding indication patients, improving the success rate of drug research and development and promoting the discovery and innovation of the new drug. The natural product, particularly the marine natural product, has the characteristics of difficult acquisition, low yield, complex structure, difficult synthesis and marking and diverse activity, and due to the diversity of the structure and the activity, the search of the action target of the natural product is one of the main bottlenecks of the research and development of new drugs, thereby greatly limiting the druggability of the natural product in China. At present, no very effective natural product target discovery method exists. The most reported methods are chemical biological methods, including affinity purification methods: the natural product can be directly linked with different mediums such as agarose beads through covalent linkage, or the natural product and biotin can be coupled through chemical reaction. The method is suitable for natural product compounds with higher activity, and the greatest disadvantage of the method is that the natural product needs to be modified, and the activity of the natural product can be damaged. The application of technologies such as light coupling and the like also increases the probability of target discovery, and many natural products are difficult to synthesize completely from the beginning, so that the application of the method is limited. The above methods also require a relatively large amount of natural products, and trace extraction of natural products does not meet the requirements of the above methods. In addition, the reported methods also include some basic biological methods, such as genetic screening method, transcriptome, phage display, yeast triple hybrid, proteomics, but these methods have difficulty in achieving wide screening of unknown target proteins. The new method reported at present is based on the activity-based protein mass spectrometry (ABPP) and the target stability of Drug Affinity Reaction (DARTS), which both greatly increase the opportunity of target identification. However, the broad-spectrum application of ABPP is greatly limited by the characteristics of being only suitable for a target with catalytic activity, covalent modification and the like; DARTS has the dependence of conformational change and the defect of being incapable of identifying low-abundance targets.

Disclosure of Invention

The invention aims to provide a method for identifying a natural product drug action target, which solves the problems that the prior art has the dependence of conformational change and can not identify low-abundance targets, and the like, so that the method can not be applied to the identification of the natural product drug action target.

In order to solve the technical problem, the technical scheme of the invention is realized as follows: the method comprises the following steps:

1) collecting a sample, and dissolving a drug to be detected in a solvent to obtain an experimental sample; taking another solvent as a control sample; respectively adding the experimental sample and the control sample into the effective cells, and incubating for 1-5h to respectively obtain a drug group sample to be detected and a solvent control group sample;

2) performing heat treatment, namely placing the drug group sample to be detected and the solvent control group sample at 30-80 ℃, performing heat treatment for 3-10min, cracking, centrifuging, and taking supernate to respectively obtain a drug protein sample to be detected and a solvent control protein sample;

3) performing electrophoresis and transfer printing, namely performing electrophoresis and transfer printing on the drug protein sample to be detected and the solvent control protein sample to obtain a drug transfer printing group to be detected and a solvent control transfer printing group;

4) dyeing, namely dyeing the drug transfer printing group to be detected and the solvent control transfer printing group by adopting a broad-spectrum protein antibody or dye, comparing, and searching for bands with protein stability differences;

5) and (3) performing mass spectrometry, namely performing electrophoresis on another drug protein sample to be detected, performing mass spectrometry on the bands with protein stability difference, and verifying to obtain a target point.

The method comprises the steps of incubating a solvent control drug and a drug to be detected with effective cells respectively to obtain suspension cells, wherein digestion is needed if adherent cells exist, and digestion is not needed if the adherent cells exist; then heat treating the cells, treating for different time at different temperature, cracking, finally performing electrophoresis and transfer printing, dyeing by using broad-spectrum molecular dye and antibody, searching for different protein band positions, cutting off adhesive tapes from corresponding positions, and performing mass spectrometry to search for action targets of the drugs. The invention relates to a natural product drug action target spot recognition method based on protein heat stability and broad spectrum molecular dyeing, which comprises an active natural product, wherein the natural product does not need to be marked in advance, and the natural product has small dosage and trace dosage, and the dosage is less than 2 mg; the protein target of the invention comprises intracellular low-abundance target molecules, has the characteristic of widely searching natural product targets, can efficiently identify potential drug targets at low cost in a label-free manner, and can promote the research of disease action mechanism and pharmacology and provide guidance information for the potential side effect of the drug and the commercialization of the drug when finding the potential drug targets. The effective cells of the invention refer to those cells which are found by activity screening in advance and have phenotype change after the action of the drug to be detected, the screening methods are the prior art, and different screening methods are available for different diseases; the method for identifying the action target of the natural product medicament is not only suitable for identifying the target of natural product active compounds and medicaments, but also comprises the target identification of active compounds and chemical medicaments from other sources.

As a preferred embodiment, in the step 4), the specific operation of dyeing is: a) preparing 5% solution of skimmed milk powder with 1 × TBST buffer solution; b) taking a drug transfer group to be detected and a solvent control transfer group, adding the solution obtained in the step a), and incubating for 1-2h at room temperature on a shaking table; c) washing with 1 × TBST buffer solution, adding protein antibody or dye, standing at 4 deg.C, and incubating overnight; d) coating an ECL luminescent reagent or a dye substrate under the condition of keeping out of the sun, and developing by adopting an imaging system; e) comparing the transfer printing group of the drug to be tested with the solvent control transfer printing group, searching for a strip with protein stability difference, and recording the molecular weight. The invention adopts the skim milk powder as the milk to distinguish specific cells from non-specific cells, and adopts the broad-spectrum protein antibody or dye to dye, so the applicability is wide, the operation is simple, the development is obvious, and the experimental effect is good; in the present invention, ECL luminescent reagent is coated for development when protein antibody is used, and dye substrate is coated for development when dye is used. TBST buffer solution is an isotonic buffer salt solution commonly used in biology, is mainly used for washing reagents such as antibodies and the like which are not specifically bound on a membrane in a Western Blot experiment, and is a ready-to-use 1 XPBST buffer solution which can be directly used. ECL chemiluminescent reagents are a new generation of Luminol-based chemiluminescent substrate reagents that are catalyzed by horseradish peroxidase (HRP) to undergo a chemical reaction that fluoresces and the result can be revealed by X-ray film sheeting and other imaging techniques or detected using a Luminometer.

In a preferred embodiment, the protein antibody is any one of a tyrosine protein kinase antibody, a serine/threonine protein kinase antibody silver stain, and biotin-labeled lectin. The protein antibody has wide application range, convenient use and good effect. The search for target proteins on cell membranes of the invention is preferably a biotin-labeled lectin antibody, of which ricin lectin RCA I is preferred.

As a preferred embodiment, in step c), when adding the protein antibody, the protein antibody is a primary antibody, and the primary antibody further comprises the following operations after incubating overnight: taking a secondary antibody, and diluting the secondary antibody by 2000-fold with 1 xTBST buffer solution; and (3) washing the transfer printing group of the drug to be detected and the solvent control transfer printing group by adopting a 1 xTBST buffer solution, adding the diluted secondary antibody into the washed transfer printing group of the drug to be detected and the solvent control transfer printing group in a shaking table at room temperature within 1-2h, slowly shaking at room temperature, and incubating for 1-2 h. When the protein antibody is selected, primary antibody is used for incubation, and then secondary antibody is used for incubation.

As a preferred embodiment, in the step 3), the specific operation of electrophoresis is: A) taking SDS-PAGE gel, wherein the SDS-PAGE gel comprises 3-5% concentrated gel and 8-12 separating gel, coating and forming pores; B) adding 8-10 mu L of a drug protein sample to be detected or a solvent control protein sample into each hole, and adding protein pre-staining Marker into the side holes; C) placing in ice water bath, performing electrophoresis at 60-80V for 20-30min, after the protein pre-staining Marker is completely separated, increasing the voltage to 120-130V, and continuing electrophoresis. The invention adopts polyacrylamide gel electrophoresis, wherein, SDS-PAGE gel is the existing widely used gel; the protein pre-staining Marker is a reagent and can be directly purchased and used. The electrophoresis operation method is simple and has good experimental effect. In the present invention, the transfer is preferably carried out by transferring the charged protein onto a Nitrocellulose (NC) film by a semi-dry transfer method, and the transfer time is about 1 to 2 hours depending on the desired molecular weight.

As a preferred embodiment, in the step 2), the specific operation of the heat treatment is: s1 enriched cells: washing the drug group sample to be detected and the solvent control group sample to respectively obtain a drug group cell to be detected and a solvent control group cell; s2 gradient heating: respectively heating the cells of the drug group to be detected and the cells of the solvent control group in a gradient manner to 30-80 ℃ to obtain cell suspension of the drug group to be detected and cell suspension of the solvent control group; s3 cell lysis: cracking the cell suspension of the drug group to be detected and the cell suspension of the solvent control group, centrifuging and collecting supernatant; s4 sample collection: adding equal volume of Loading Buffer into the supernatant respectively, resuspending, decocting in boiling water for 15-20min, standing at-20 deg.C, and storing. The invention adopts a gradient heating method for heat treatment, and the Loading Buffer is a sample Loading Buffer solution and mainly has two functions, namely, the indicator bromophenol blue and the xylene cyanide FF play a role in indicating and display the progress of electrophoresis so as to stop the electrophoresis at the right time; second, the component glycerol increases the density of the sample to a level greater than the TAE, thereby settling into the wells and preventing the sample from drifting out of the wells.

In a preferred embodiment, the lysis is any one of repeated freeze thawing with liquid nitrogen, lysis with cell lysate and ultrasonic disruption lysis. The cracking method of the invention has various cracking modes, wherein when liquid nitrogen is adopted for repeated freeze thawing cracking, repeated freeze thawing is usually required for three times; of course, lysis with cell lysates or sonication can also be used.

In a preferred embodiment, in step S2, the cells of the drug to be tested and the cells of the solvent control group are heated to 40-60 ℃ in a gradient manner. In the heat treatment process of the present invention, the temperature may preferably be 40-60 ℃, and most proteins are unstable in this temperature range, while Tubulin as an internal reference may still be detected.

As a preferred embodiment, in step 1), the specific operation of sample collection is: sa cell plating: digesting and counting effective cells in good growth state and logarithmic growth phase with pancreatin, inoculating, and inoculating at 37 deg.C and 5% CO₂Culturing for 24h under the condition; adding Sb cells: setting the experimental sample and the control sample, respectively, and adding the effective cells cultured in step Sa at 37 deg.C and 5% CO₂Continuously culturing for 1-3h under the condition; sc-collected cells: removing the culture medium, adding cold PBS, washing, adding pancreatin for digestion, and uniformly blowing to make all adherent cells suspended. The sample collection is obtained by adding medicine into the cell seed plate and the cells and collecting the cells, only the adherent cells are digested in the digestion process, and the non-adherent cells do not need to be digested; the cold PBS is a precooled PBS buffer solution, and the PBS buffer solution is phosphate buffer saline (phosphate buffer saline) which is generally used as a solvent and plays a role in dissolving a protective reagent; it is a buffer solution which is most widely used in biochemical research, and the main component of the buffer solution is Na₂HPO₄、KH₂PO₄NaCl and KCl.

As a preferred embodiment, in the step 5), the specific operation of mass spectrometry is: and (3) taking a drug protein sample to be detected, carrying out electrophoresis according to the step 3), wherein the sample loading amount during electrophoresis is 20-30 mu L, cutting off adhesive strips near the difference, rinsing with ultrapure water, and carrying out mass spectrometry. The invention obtains the protein with the molecular weight of the protein stability difference by mass spectrometry, combines with the research results of some cell signal channels, verifies and locks the target protein, thereby determining the target point.

Compared with the prior art, the invention has the beneficial effects that: the invention relates to a natural product drug action target recognition method based on protein heat stability and broad spectrum molecular dyeing, wherein the natural product drug comprises an active natural product, the natural product does not need to be marked in advance, and the natural product has small dosage, trace dosage and dosage less than 2 mg; the protein target of the invention comprises intracellular low-abundance target molecules, has the characteristic of widely searching natural product targets, can efficiently identify potential drug targets at low cost in a label-free manner, and can promote the research of disease action mechanism and pharmacology and provide guidance information for the potential side effect of the drug and the commercialization of the drug when finding the potential drug targets.

Drawings

FIG. 1 is a process flow diagram of a method for identifying a natural product drug action target according to the present invention;

FIG. 2 is a graph showing the results of Muriceidine A-4 inhibiting proliferation of various tumor cells;

FIG. 3 is a graph showing the effect of Muriceidine A-4 on the proliferation inhibition of MDA-MB-231 cells;

FIG. 4 is a graph showing the effect of Muriceidine A-4 on a portion of the signaling protein in MDA-MB-231 cells;

FIG. 5 is a graph of the binding of Muriceidine A-4 to a glycoprotein in MDA-MB-231 cells;

FIG. 6 is a graph of the binding of Muriceidine A-4 to transferrin receptor in MDA-MB-231 cells;

FIG. 7 shows FeSO₄Effect on Muriceidine A-4 induced apoptosis of MDA-MB-231 cellsA drawing;

FIG. 8 shows FeSO₄Results of the effect on Muriceidine A-4 induced MDA-MB-231 apoptosis are shown.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to the attached figure 1, the method for identifying the action target of the natural product medicament comprises the following steps:

1) collecting a sample, and dissolving a drug to be detected in a solvent to obtain an experimental sample; taking another solvent as a control sample; respectively adding the experimental sample and the control sample into the effective active cells, and incubating for 1-5h to respectively obtain a drug group sample to be detected and a solvent control group sample;

2) performing heat treatment, namely placing the drug group sample to be detected and the solvent control group sample at 30-80 ℃, performing heat treatment for 3-10min, cracking, centrifuging, and taking supernate to respectively obtain a drug protein sample to be detected and a solvent control protein sample;

3) performing electrophoresis and transfer printing, namely performing electrophoresis and transfer printing on the drug protein sample to be detected and the solvent control protein sample to obtain a drug transfer printing group to be detected and a solvent control transfer printing group;

4) dyeing, namely dyeing the drug transfer printing group to be detected and the solvent control transfer printing group by adopting a broad-spectrum protein antibody or dye, comparing, and searching for bands with protein stability differences;

5) and (3) performing mass spectrometry, namely performing electrophoresis on another drug protein sample to be detected, performing mass spectrometry on the bands with protein stability difference, and verifying to obtain a target point.

Preferably, in the step 4), the dyeing operation is as follows: a) preparing 5% solution of skimmed milk powder with 1 × TBST buffer solution; b) taking a drug transfer group to be detected and a solvent control transfer group, adding the solution obtained in the step a), and incubating for 1-2h at room temperature on a shaking table; c) washing with 1 × TBST buffer solution, adding protein antibody or dye, standing at 4 deg.C, and incubating overnight; d) coating an ECL luminescent reagent or a dye substrate under the condition of keeping out of the sun, and developing by adopting an imaging system; e) comparing the transfer printing group of the drug to be tested with the solvent control transfer printing group, searching for a strip with protein stability difference, and recording the molecular weight.

Further, the protein antibody is any one of a tyrosine protein kinase antibody, a serine/threonine protein kinase antibody, a protein silver stain and biotin-labeled lectin.

Specifically, in the step c), when the protein antibody is added, the protein antibody is a primary antibody, and the primary antibody further comprises the following operations after the overnight incubation: taking a secondary antibody, and diluting the secondary antibody by 2000-fold with 1 xTBST buffer solution; and (3) washing the transfer printing group of the drug to be detected and the solvent control transfer printing group by adopting a 1 xTBST buffer solution, adding the diluted secondary antibody into the washed transfer printing group of the drug to be detected and the solvent control transfer printing group in a shaking table at room temperature within 1-2h, slowly shaking at room temperature, and incubating for 1-2 h.

More preferably, in the step 3), the electrophoresis specifically operates as follows: A) taking SDS-PAGE gel, wherein the SDS-PAGE gel comprises 3-5% concentrated gel and 8-12 separating gel, coating and forming pores; B) adding 8-10 μ L of drug protein sample to be detected or solvent control protein sample into each hole, and adding protein pre-staining Marker into the side holes; C) placing in ice water bath, performing electrophoresis at 60-80V for 20-30min, after the protein pre-staining Marker is completely separated, increasing the voltage to 120-130V, and continuing electrophoresis.

Further, in the step 2), the specific operation of the heat treatment is as follows: s1 enriched cells: washing the drug group sample to be detected and the solvent control group sample to respectively obtain a drug group cell to be detected and a solvent control group cell; s2 gradient heating: heating the cells of the drug group to be detected and the cells of the solvent control group to 30-80 ℃ in a gradient way respectively, and obtaining cell suspension of the drug group to be detected and cell suspension of the solvent control group; s3 cell lysis: cracking the cell suspension of the drug group to be detected and the cell suspension of the solvent control group, centrifuging and collecting supernatant; s4 sample collection: adding equal volume of Loading Buffer into the supernatant respectively, resuspending, decocting in boiling water for 15-20min, standing at-20 deg.C, and storing.

More specifically, the lysis is any one of repeated freeze thawing of liquid nitrogen, lysis of cell lysate and ultrasonic disruption lysis.

Preferably, in step S2, during the gradient heating, the cells of the drug group to be tested and the cells of the solvent control group are heated to 40-60 ℃ respectively by gradient heating.

Still further, in the step 1), the specific operation of collecting the sample is as follows: sa cell plating: digesting and counting effective cells in good growth state and logarithmic growth phase with pancreatin, inoculating, and inoculating at 37 deg.C and 5% CO₂Culturing for 24h under the condition; adding Sb cells: setting the experimental sample and the control sample, respectively, and adding the effective cells cultured in step Sa at 37 deg.C and 5% CO₂Continuously culturing for 1-3h under the condition; sc-collected cells: removing the culture medium, adding cold PBS, washing, adding pancreatin for digestion, and uniformly blowing to make all adherent cells suspended.

More specifically, in the step 5), the mass spectrometry includes: and (3) taking a drug protein sample to be detected and a solvent control protein sample, carrying out electrophoresis according to the step 3), wherein the sample loading amount during electrophoresis is 20-30 mu L, cutting off adhesive strips near the difference, rinsing with ultrapure water, and carrying out mass spectrometry.