A method for developing a protac for identifying a crl complex based on arih1 mass spectrometry

Abstract

The application discloses a PROTAC research and development method for identifying CRL complex based on ARIH1 mass spectrum, and relates to the technical field of biology; and comprises the following steps: S1, constructing a PBTE-C357 SARIH1 overexpressed H9 cell line; S2, inducing the H9 stem cell line to differentiate from ESC to mesoderm and endoderm; S3, overexpressing ARIH1 immunoprecipitation experiment; S31, inoculating the H9 cells into 12 15cm cell dishes respectively, adding 20mL RPMI culture medium into each cell dish, and simultaneously adding Doxycycline to induce FLAG-ARIH1 overexpression; and preparing for sampling after overexpression. The application is based on the characteristics that ARIH1 serves as a CO-E3 ligase and forms a stable complex with activated CRL that occurs in a pseudo-substance, and uses TMT-IP / MS technology to accurately identify the CRL substrate receptor in an activated state.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a PROTAC development method based on ARIH1 mass spectrometry identification of CRL complex. BACKGROUND

[0002] The Cullin-RING ubiquitin ligase (CRL) family is the largest E3 ubiquitin ligase family in the ubiquitin-proteasome system, which regulates protein degradation by recognizing specific substrate receptors. CRL family plays a key role in various biological processes, including cell cycle regulation, signal transduction and cancer development. However, due to the large number of CRL family members and complex functions, identifying the substrate receptors of the activated state has been a research difficulty.

[0003] TMT-IP / MS (Tandem Mass Tag immunoprecipitation mass spectrometry) is an advanced method combining TMT polypeptide labeling technology and immunoprecipitation mass spectrometry, which is widely used in protein interaction research, especially in PROTAC (Proteolysis Targeting Chimera) development. TMT (Tandem Mass Tag) technology is a kind of polypeptide in vitro labeling technology developed by Thermo Scientific Company in the United States. This technology labels the amino group of polypeptide by specific labeling, and uses isotope labels to realize the quantitative analysis of polypeptide in mass spectrometry analysis. TMT technology has the characteristics of high sensitivity, high throughput and high accuracy, and can simultaneously quantitatively analyze multiple samples, which is suitable for complex proteomics research. IP / MS (immunoprecipitation-mass spectrometry combined technology) is a protein interaction research method based on immunoprecipitation and mass spectrometry analysis. Its principle is to capture target proteins and their interacting proteins by specific antibodies, and then enrich them by immunoprecipitation, and then identify these proteins by mass spectrometry analysis. TMT-IP / MS combines the quantitative ability of TMT and the protein interaction analysis ability of IP / MS. By TMT labeling technology, the polypeptides after immunoprecipitation are quantitatively analyzed, which can more accurately identify and quantify protein interactions. TMT-IP / MS technology has important application value in PROTAC development with its high specificity, high sensitivity and quantitative analysis ability, which can provide strong support for targeted protein degradation research.

[0004] In existing studies, the teams of Sun Lei and Chen Zhenguo used cryo-electron microscopy to reveal the dynamic assembly and molecular mechanisms of the CRL3 complex in multiple states during its catalytic cycle. They studied the self-assembly, substrate recruitment, activation, deactivation, and substrate-receptor exchange processes of the CRL3KBTBD2 complex and analyzed its structure in different states. In addition, Professor Xu Chao's research group studied the assembly pattern of the CRL2FEM1B complex and the molecular mechanism of its substrate recognition. These studies provide an important foundation for understanding the functions of the CRL family, but they mainly focus on structural analysis and mechanism research, and only on a certain substrate of CRL. They have not yet involved the use of TMT-IP / MS technology to systematically identify multiple substrate receptors in the activated state.

[0005] Compared with traditional small molecules, PROTAC molecules are more difficult to develop, and the bottlenecks mainly focus on drug development, synthesis process, evaluation methods, etc. The development bottlenecks of PROTAC in new drug research and development include the following:

[0006] ① Druggability issues caused by large molecular weight: Traditional small molecule drugs generally conform to the druggability principle - Lipinski's "Five Rules of Drug-Like Properties", but PROTAC molecules often have a large molecular weight. The molecular weight of currently reported PROTACs is mostly above 700 Daltons. Therefore, compared with traditional small molecule inhibitors, PROTAC molecules have limited water solubility and cell permeability. Poor pharmacokinetic properties are a major obstacle to their druggability.

[0007] ② The number of available E3 ubiquitin ligases is limited: As one of the components of PROTAC, E3 ubiquitin ligases plays a vital role. Although studies have found that more than 600 E3 ligases can function in human cells, most of these E3 ligases have limited tissue distribution. Even the most widely used E3 ligases, CEBN, MDM2, and XIAP, still have a small number of tissues that are not covered. For example, MDM2 ligase is present in 99% of test samples, while CRBN is only expressed in 76% of test samples. Therefore, there is still much room for development of E3 ligases with broad tissue distribution and ligands with good druggable properties. Of the more than 600 E3 ligases, only 12 have been used in PROTACs, two of which have entered clinical trials, and the other 10 are in the preclinical exploration stage, currently mainly targeting the CRBN E3 ubiquitin ligase.

[0008] ③ When high concentrations of PROTACs tend to form binary complexes rather than ternary complexes in the presence of abundant E3 ligases and targets, the Hook effect inevitably occurs, which brings difficulties to the rational design of in vivo dosage.

[0009] CRL (Cullin RING ubiquitin ligase) family is the largest E3 ubiquitin ligase superfamily, containing more than 200 family members, regulating ubiquitination and degradation of about 20% of proteins in cells. As the most important E3 superfamily, CRL has a precise regulatory system for its biochemical activity: CRL is a modular complex enzyme, and its Cullin protein can assemble hundreds of different substrate receptors, thereby ubiquitinating specific substrate proteins. The substrate proteins of CRL include Cyclin D, HIF1a, AKT, Nrf2, PCNA, PD-1, PD-L1 and other key proteins, thus playing an important regulatory role in cell cycle, signal transduction, DNA damage repair, immune checkpoints and other biological processes. The expression or activity of various CRL family members is abnormal in the occurrence of diseases such as tumors, and is an important therapeutic target; in addition, for example, CRL4CRBN and CRL2VHL are E3 ubiquitin ligases widely used in PROTAC technology, and the successful approval of ARV-110 based on CRL4CRBN shows that CRL has great potential and space in this emerging new drug development field. Although CRL has important biological functions and plays a key regulatory role in various diseases, the development of small molecule drugs targeting CRL ubiquitin ligase is far behind that of kinases that catalyze phosphorylation modification. One of the main reasons is that the research on the regulatory mechanism and function of CRL family is relatively lacking.

[0010] According to the fact that ARIH1 can form a stable complex with CO-E3 and activated CRL (Cullin-RING ubiquitin ligase) that ubiquitinates, the present application mainly identifies the CRL substrate receptor (Substrate Receptor) in the activated state based on TMT-IP / MS (Tandem Mass Tag immunoprecipitation mass spectrometry) of ARIH1, and screens and identifies CRL family members with key biological functions. SUMMARY

[0011] The purpose of the present application is to solve the problems existing in the prior art and provide a PROTAC research and development method based on ARIH1 mass spectrometry identification of CRL complex.

[0012] In order to achieve the above purpose, the present application adopts the following technical scheme:

[0013] A PROTAC research and development method based on ARIH1 mass spectrometry identification of CRL complex, comprising the following steps:

[0014] S1: Constructing a PBTE-C357S ARIH1 overexpressed H9 cell line;

[0015] S2: Induce the H9 stem cell line to differentiate from ESC to mesoderm and endoderm;

[0016] S3: Immunoprecipitation experiment of overexpressed ARIH1;

[0017] S31: H9 cells were seeded into 12 15 cm cell dishes, and 20 mL of RPMI medium was added. Doxycycline was also added to induce FLAG-ARIH1 overexpression. After overexpression, samples were prepared for harvest. On the day of harvest, MLN was added to the control group.

[0018] S32: Use MCLB buffer containing protease inhibitors to lyse cells. Before the start of lysis, add protease inhibitors to the lysis buffer and place the lysis buffer containing protease inhibitors on ice. Add lysis buffer to each dish, scrape the cells and collect them into tubes, rotate on a shaker, and then centrifuge in a low-temperature centrifuge.

[0019] S33: Combine the supernatants from the two tubes into one sample, filter it through a filter, and then filter it into the tube containing FLAGBeads and rotate it on a shaker.

[0020] S34: Transfer the sample and FLAG Beads mixture to a tube, wash with MCLB buffer, elute with 3× FLAG peptide, and combine the eluates into an EP tube;

[0021] S35: Add TCEP to the sample and reduce for 1 hour, then alkylate with iodoacetamide; precipitate with TCA / acetonitrile, resuspend in buffer, add LysC to the resuspension, and incubate at room temperature overnight;

[0022] S36: The next day, trypsin was added and digested for 6 hours, followed by acetonitrile (ACN) and reaction at room temperature. TMT was added and reaction was continued at room temperature.

[0023] S37: Cells differentiated into mesoderm and endoderm were treated in the same manner. Then, 2 μL of each sample was taken and combined with 100 μL of 5% FA / 5% ACN. The cells were treated with a stage tip and dried before mass spectrometry analysis.

[0024] Preferably, in S1, the construction of the PBTE-C357SARIH1 overexpressing H9 cell line is as follows:

[0025] S11: One day before transfection, seed H9 cells in a 6-well plate and add 2 mL of medium without antibiotics to ensure that the cells are 70%-90% confluent at the time of transfection.

[0026] S12: Prepare plasmid DNA and transfection reagents. Calculate the required amounts of PBTE-C357SARIH1 and hypbase plasmids and mix them in a 3:1 ratio.

[0027] S13: Add 100 μL of serum-free Opti-MEM medium to a sterile centrifuge tube, then add 2 μg of plasmid DNA and mix well. Add 100 μL of serum-free Opti-MEM medium to another sterile centrifuge tube, then add 5 μL of Lipofectamine 2000, mix well and let stand at room temperature for 5 minutes. Add the DNA dilution to the Lipofectamine 2000 dilution, mix well, and let stand at room temperature for 20 minutes to form a DNA-Lipofectamine 2000 complex. Slowly add the prepared DNA-Lipofectamine 2000 complex to the cell culture medium in each well, and shake the culture plate to ensure even distribution of the complex.

[0028] S14: Place the culture plate in the incubator and continue culturing. After transfection, replace the culture medium with serum-containing complete medium and continue culturing.

[0029] S15: Transfection: After a set time, change the medium to complete medium containing serum and add hygromycin B for selection. When more than 80% of the control cells die, remove the antibiotics from the experimental group and replace with complete medium containing serum, and wait for the state to return to normal;

[0030] S16: After the stably transfected cells can be passaged normally, separate the wells and perform WB to detect the expression of ARIH1.

[0031] Preferably, in said S14, specifically: placing the culture plate in an incubator at 37° C. and 5% CO 2 for continued culture; 6 hours after transfection, replacing the culture medium with serum-containing complete culture medium, and continuing culture for 48 hours.

[0032] Preferably, in said S15, specifically: 48 hours after transfection, the medium is changed to a complete medium containing serum, and 100 μg / ml of hygromycin B is added for screening. When more than 80% of the control cells die, the antibiotics of the experimental group are removed, and the medium is replaced with a complete medium containing serum, and the cells are allowed to return to normal.

[0033] Preferably, in said S2, the induction of H9 stem cell line differentiation from ESC to mesoderm and endoderm is specifically as follows:

[0034] S21: When the ARIH1-overexpressing cells grow to confluence, they are plated;

[0035] S22: On the second day, replace the culture medium with Advanced RPMI 1640 medium and add the following reagents per ml:

[0036] Glutamax (100×): 10 μL;

[0037] CHIR99021 (5 μM): 1 μL;

[0038] 200ng / mL Activin A: 0.2μL;

[0039] From this time on, doxycycline (0.5 mg / ml) was added simultaneously to induce FLAG-ARIH1 overexpression;

[0040] S23: On the third day, the medium was changed using the same medium as in S22;

[0041] S24: Day 4, at which point the cells have differentiated into the mesoderm, and the subsequent induction medium is prepared and the medium is changed;

[0042] S25: Day 5, cells differentiate into endoderm stem cells.

[0043] Preferably: In the S21, for a 6-well plate, 1×10 5 After plating, Y27632 was added at a dilution of 1000:1 to inhibit cell apoptosis.

[0044] Preferably, in the S24, Advanced RPMI 1640 medium is used, and the following reagents are added per ml:

[0045] Glutamax (100×): 10 μL;

[0046] 100ng / mL Activin A: 1μL.

[0047] Preferably, in the S31, H9 cells were cultured at 2.7×10 7 The cells were seeded in 12 15 cm cell dishes at a density of 100 cells / mL and divided into 6 experimental groups and 6 control groups;

[0048] Doxycycline was added at a concentration of 0.5 mg / ml; FLAG-ARIH1 overexpression was induced, and samples were collected 48 hours after overexpression. On the day of collection, MLN was added to the 15 cm dish of the control group at 1 μM for 6 hours;

[0049] In the S32, 1.6 mL of lysis buffer was added to each dish, cells were scraped and collected into a tube, rotated on a shaker at 4°C for 10 minutes, and then centrifuged at 1200 rpm in a 4°C low-temperature centrifuge for 15 minutes.

[0050] Preferred: in S33, the supernatants of the two tubes are combined into one sample and filtered using a 0.45 μm filter, and then filtered into a tube containing 60 μL of FLAG Beads and rotated on a 4℃ shaker for 3 hours;

[0051] In S34, the sample is transferred to a tube with the FLAG Beads mixture, washed 3 times with MCLB buffer for 15 minutes each time; next, eluted using 3x FLAG peptide, 200 μL each time, a total of 2 times, and the eluate is combined into one EP tube.

[0052] Preferred: in S35, TCEP is added to the sample at a final concentration of 10 mM, reduced at 37℃ for 1 hour, and then the sample is alkylated using iodoacetamide at a final concentration of 15 mM; precipitated using TCA / acetonitrile, and then resuspended in 50 μL of 0.2M EPPS pH 8.0 buffer, 1 μL of LysC is added to the resuspension, and incubated at room temperature overnight;

[0053] In S36, 1 μL of trypsin is added, and incubated at 37℃ for 6 hours, and then 15 μL of acetonitrile (ACN) is added, and incubated at room temperature for 10 minutes; 4 μL of TMT is added, and incubated at room temperature for 1.5 hours.

[0054] The beneficial effects of the present application are:

[0055] 1. The present application is based on the characteristics of ARIH1 as a CO-E3 ligase forming a stable complex with CRL activated by neusylation, and uses TMT-IP / MS technology to accurately identify CRL substrate receptors in an activated state.

[0056] 2. The technology of the present application can be applied to different cell lines, including various tumor cell lines, which helps to systematically study the dynamic assembly of the CRL family in the occurrence and development of cancer; by identifying CRL family members with key biological functions, it provides CRL family members with target significance for the research and development of new PROTACs.

[0057] 3. The technology of the present application can quickly identify key CRL substrate receptors, providing clear target points for the design of PROTACs, thereby shortening the period from target point discovery to drug design; through accurate screening and identification, the investment in ineffective target points is reduced, and the research and development cost is reduced.

[0058] 4. The technology of the present application is suitable for a variety of cell lines, including different tumor cell lines, and can systematically study the dynamic assembly and functional regulation of CRL family in different cancers; this provides the possibility for developing personalized PROTACs for specific tumor types; through application in different tumor cell lines, CRL substrate receptors with tumor specificity can be found, thereby developing more precise tumor treatment drugs.

[0059] 5. Traditional small molecule drugs are difficult to target "undruggable" proteins such as transcription factors and scaffold proteins; the CRL substrate receptors identified by the TMT-IP / MS technology of the present application provide the possibility for developing PROTACs for these "undruggable" targets; each PROTAC molecule can degrade multiple target protein molecules, has catalytic degradation function, and therefore can exert significant drug efficacy with low dosage and long-lasting drug efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 A technical idea diagram of a PROTAC development method based on ARIH1 mass spectrometry identification of CRL complexes is proposed for the present application;

[0061] Figure 2 A volcano plot analysis result diagram of the influence of ARIH1 overexpression and MLN4924 treatment on substrate receptor activity in human embryonic stem cells is provided for the present application;

[0062] Figure 3 A volcano plot analysis result diagram of the influence of ARIH1 overexpression and MLN4924 treatment on substrate receptor activity in human mesoderm cells is provided for the present application;

[0063] Figure 4 A volcano plot analysis result diagram of the influence of ARIH1 overexpression and MLN4924 treatment on substrate receptor activity in human endoderm cells is provided for the present application;

[0064] Figure 5 A schematic diagram of the activity state of multiple substrate receptors in different cell types is provided for the present application. DETAILED DESCRIPTION

[0065] The technical solutions of the present application will be further described in detail below in combination with specific embodiments.

[0066] Example 1:

[0067] A PROTAC development method based on ARIH1 mass spectrometry identification of CRL complexes includes the following experimental steps:

[0068] S1: Construct PBTE-C357S ARIH1 overexpressing H9 cell line:

[0069] S11: One day before transfection, H9 cells were inoculated in 6-well plates at an appropriate density (about 0.5-2 x 10 5 cells per well) with 2 mL of culture medium without antibiotics to ensure that the confluence of the cells reached 70-90% at the time of transfection for optimal transfection efficiency;

[0070] S12: Prepare plasmid DNA and transfection reagent, calculate the amount of PBTE-C357S ARIH1 and hypbase plasmid required, and mix them in a ratio of 3:1;

[0071] S13: Add 100 μL of serum-free Opti-MEM medium (Gibco) to a sterile centrifuge tube, then add 2 μg of plasmid DNA (PBTE-C357S ARIH1 and hypbase plasmid mixed in a ratio), mix gently, add 100 μL of serum-free Opti-MEM medium to another sterile centrifuge tube, then add 5 μL of Lipofectamine 2000 (Thermo Scientific), mix gently, and let stand at room temperature for 5 minutes. Add the DNA dilution to the Lipofectamine 2000 dilution, mix gently, and let stand at room temperature for 20 minutes to form a DNA-Lipofectamine 2000 complex. Slowly add the prepared DNA-Lipofectamine 2000 complex to the cell culture medium in each well, and gently shake the culture plate to distribute the complex evenly;

[0072] S14: Place the culture plate in a 37°C, 5% CO2 incubator and continue to culture. After 6 hours of transfection, replace the medium with complete medium containing serum and continue to culture for 48 hours;

[0073] S15: After 48 hours of transfection, replace the medium with complete medium containing serum and add Hygromycin B (sangon, 100 μg / ml) for selection. When the control cells are more than 80% dead, remove the antibiotic from the experimental group and replace it with complete medium containing serum. Wait for the state to return to normal.

[0074] S16: After the stable transfection cells can be normally passaged, the expression of ARIH1 is detected by WB (detected by FLAG tag antibody), compared with the control, the experimental group appears obvious overexpression band (~ 70kDa), which indicates that the stable cell line is successfully constructed.

[0075] S2: Induce H9 stem cell line to differentiate from ESC (Embryonic Stem Cells) to Mesendoderm (Mesendoderm) and Endoderm (Endoderm):

[0076] S21: When the cells overexpressing ARIH1 grow to confluence, plate them. For a 6-well plate, plate 1×10 5 After plating, Y27632 (MCE, 1000:1 dilution) was added to inhibit cell apoptosis;

[0077] S22: On the second day, the culture medium was replaced (configuration as follows: Advanced RPMI 1640 medium, with the following reagents added per ml: Glutamax (100×) 10 μL, CHIR99021 (5 μM) 1 μL, 200 ng / mL Activin A 0.2 μL). From this point on, Doxycycline (0.5 mg / ml) was added to induce FLAG-ARIH1 overexpression;

[0078] S23: On the third day, the medium was changed using the above-mentioned culture medium;

[0079] S24: Day 4, at which point the cells have differentiated into mesendoderm, and the subsequent induction medium is prepared and the medium is changed (Advanced RPMI 1640 medium is used, and the following reagents are added per ml: Glutamax (100×) 10 μL, 100 ng / mL Activin A 1 μL);

[0080] S25: On day 5, cells have successfully differentiated into endoderm stem cells. Subsequent experiments can be performed.

[0081] S3: Overexpression of ARIH1 immunoprecipitation (Co-IP) experiment:

[0082] S31: H9 cells were cultured at an appropriate density (2.7×10 7 Cells were seeded into 12 15-cm cell dishes (6 in the experimental group and 6 in the control group), and 20 mL of RPMI medium (Gibco) was added. Doxycycline (0.5 mg / ml) was also added to induce FLAG-ARIH1 overexpression. Samples were collected 48 hours after overexpression. On the day of collection, MLN (1 μM) (MLN is a pseudotype inhibitor that can inactivate CRL) was added to the 15-cm dish of the control group for 6 hours.

[0083] S32: Lyse cells using MCLB buffer (Modified Cell Lysis Buffer: Tris 0.5 g, NaCl 0.5 g, SDS 0.1 g, Triton-X-100 1 mL, deionized water to 10 mL, adjust pH to 7.4 with HCl) containing protease inhibitors. Before lysis, add protease inhibitors (MCE) to the lysis buffer. Place the lysis buffer containing protease inhibitors on ice. Add 1.6 mL of lysis buffer to each dish, scrape the cells and collect them in a 2 mL tube. Rotate on a shaker at 4°C for 10 min, then centrifuge at 12,000 rpm in a 4°C refrigerated centrifuge for 15 min.

[0084] S33: The supernatants from the two tubes were combined into one sample and filtered using a 0.45 μm filter. The sample was then filtered into a 5 mL tube containing 60 μL of FLAGBeads (previously washed with PBST) and rotated on a shaker at 4°C for 3 h.

[0085] S34: Transfer the sample and FLAG beads mixture to a 15 mL tube and wash three times with MCLB buffer for 15 minutes each. Next, elute with 3× FLAG peptide (Cytiva) twice, 200 μL each time, and combine the eluates into a 1.5 mL EP tube.

[0086] S35: Add TCEP to a final concentration of 10 mM to the sample and reduce at 37°C for 1 hour. Then, alkylate the sample with iodoacetamide to a final concentration of 15 mM. Precipitate with TCA / acetonitrile and resuspend in 50 μL 0.2 M EPPS pH 8.0 buffer. Add 1 μL LysC (Wako Chemicals) to the resuspension and incubate at room temperature overnight.

[0087] S36: The next day, add 1 μL of trypsin (Gibco) and digest at 37°C for 6 hours. Then add 15 μL of acetonitrile (ACN) and react at room temperature for 10 minutes. Add 4 μL of TMT (Thermo Scientific) and react at room temperature for 1.5 hours.

[0088] S37: The above operation was also performed on cells differentiated into mesoderm and endoderm. Then, 2 μL was taken from each sample, combined with 100 μL 5% FA / 5% ACN, and treated with Stage tip. After drying, mass spectrometry analysis was performed.

[0089] like Figures 2-4The figure shows the results of a volcano plot analysis of the effects of ARIH1(C357S) overexpression and MLN4924 treatment on substrate receptor activity in different cell types. The figure includes three cell types: hESC (human embryonic stem cells), hMes (human mesoderm cells), and hEndo (human endoderm cells). In each graph, the horizontal axis represents the log2 fold change of the substrate receptor, and the vertical axis represents the -log10 p-value, which is used to measure statistical significance. It can be seen that in the experimental group without MLN, the number of substrate receptors in the activated state that assembled into complexes with ARIH1 increased to varying degrees at different cell stages, compared to the negative control group with MLN.

[0090] like Figure 5 As shown in the figure, this heat map shows the activity status of multiple substrate receptors in different cell types. The figure includes three cell types: embryonic stem cells (ESC), mesoderm (Mes), and endoderm (Endo). Dark squares indicate that the substrate receptor is not detected (ND) or inactive (Inactive) in a specific cell type, while light squares indicate that the substrate receptor is active (Active) in a specific cell type. This helps to understand the dynamic changes and functional differences of CRL complex substrate receptors in different cell types.

[0091] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A PROTAC development method based on ARIH1 mass spectrometry identification of CRL complexes, characterized in that: The steps include: S1: Construction of PBTE-C357S ARIH1-overexpressing H9 cell line; S2: Induce the H9 stem cell line to differentiate from ESC to mesoderm and endoderm; S3: Immunoprecipitation experiment of overexpressed ARIH1; S31: H9 cells were seeded into 12 15 cm cell dishes, and 20 mL of RPMI medium was added. Doxycycline was also added to induce FLAG-ARIH1 overexpression. After overexpression, samples were prepared for harvest. On the day of harvest, MLN was added to the control group. S32: Lyse cells using MCLB buffer containing protease inhibitors. Before lysis begins, add protease inhibitors to the lysis buffer and place the lysis buffer containing protease inhibitors on ice. Add lysis buffer to each dish, scrape the cells and collect them into a tube, rotate on a shaker, and then centrifuge using a refrigerated centrifuge. S33: Combine the supernatants from the two tubes into one sample, filter it through a filter, and then filter it into the tube containing FLAGBeads and rotate it on a shaker. S34: Transfer the sample and FLAG Beads mixture to a tube, wash with MCLB buffer, elute with 3× FLAG peptide, and combine the eluates into an EP tube; S35: Add TCEP to the sample and reduce for 1 hour, then alkylate with iodoacetamide; precipitate with TCA / acetonitrile, resuspend in buffer, add LysC to the resuspension, and incubate at room temperature overnight; S36: The next day, trypsin was added and digested for 6 hours, followed by acetonitrile (ACN) and reaction at room temperature. TMT was added and reaction was continued at room temperature. S37: Cells differentiated into mesoderm and endoderm were treated in the same manner. 2 µL of each sample was then combined in 100 µL of 5% FA / 5% ACN, treated with a Stage Tip, and dried before mass spectrometry analysis.

2. A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry according to claim 1, characterized in that In S1, the construction of the PBTE-C357SARIH1 overexpressing H9 cell line is as follows: S11: One day before transfection, seed H9 cells in a 6-well plate and add 2 mL of medium without antibiotics to ensure that the cells are 70%-90% confluent at the time of transfection. S12: Prepare plasmid DNA and transfection reagents. Calculate the required amounts of PBTE-C357SARIH1 and hypbase plasmids and mix them in a 3:1 ratio. S13: Add 100µL serum-free Opti-MEM medium to a sterile centrifuge tube, then add 2µg plasmid DNA and mix well. Add 100µL serum-free Opti-MEM medium to another sterile centrifuge tube, then add 5µL Lipofectamine 2000, mix well and let stand at room temperature for 5 minutes. Add the DNA dilution to the Lipofectamine 2000 dilution, mix well, and let stand at room temperature for 20 minutes to form a DNA-Lipofectamine 2000 complex. Slowly add the prepared DNA-Lipofectamine 2000 complex to the cell culture medium of each well and shake the culture plate to evenly distribute the complex. S14: Place the culture plate in the incubator and continue culturing. After transfection, replace the culture medium with serum-containing complete medium and continue culturing. S15: Transfection: After a set time, change the medium to complete medium containing serum and add hygromycin B for selection. When more than 80% of the control cells die, remove the antibiotics from the experimental group and replace with complete medium containing serum, and wait for the state to return to normal; S16: After the stably transfected cells can be passaged normally, separate the wells and perform WB to detect the expression of ARIH1.

3. A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry according to claim 2, characterized in that In the S14, specifically, the culture plate is placed in an incubator at 37° C. and 5% CO 2 for continued culture. Six hours after transfection, the culture medium is replaced with a complete medium containing serum and culture is continued for 48 hours.

4. A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry according to claim 3, characterized in that In S15, specifically, 48 hours after transfection, the medium was changed to complete medium containing serum, and 100 μg / ml of hygromycin B was added for screening. When more than 80% of the control cells died, the antibiotics in the experimental group were removed, and the medium was replaced with complete medium containing serum, and the cells were allowed to return to normal.

5. A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry according to claim 4, characterized in that In S2, the H9 stem cell line is induced to differentiate from ESC to mesoderm and endoderm as follows: S21: When the ARIH1-overexpressing cells grow to confluence, they are plated; S22: On the second day, replace the culture medium with Advanced RPMI 1640 medium and add the following reagents per ml: 100×Glutamax: 10µL; 5µM CHIR99021: 1µL; 200 ng / mL Activin A: 0.2µL; From this time on, doxycycline (0.5 mg / ml) was added simultaneously to induce FLAG-ARIH1 overexpression; S23: On the third day, the medium was changed using the same medium as in S22; S24: Day 4, at which point the cells have differentiated into the mesoderm, and the subsequent induction medium is prepared and the medium is changed; S25: Day 5, cells differentiate into endoderm stem cells.

6. A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry according to claim 5, characterized in that In the S21, 1×10 5 cells were plated per well of a 6-well plate. After plating, Y27632 was added at a dilution of 1000:1 to inhibit cell apoptosis.

7. A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry according to claim 5, characterized in that In the S24, Advanced RPMI 1640 medium was used, and the following reagents were added per ml: 100×Glutamax: 10µL; 100 ng / mL Activin A: 1µL.

8. A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry according to claim 1, characterized in that In the S31, H9 cells were seeded at a density of 2.7×107 cells in 12 15 cm cell dishes and divided into 6 experimental groups and 6 control groups; Doxycycline was added at a concentration of 0.5 mg / ml; FLAG-ARIH1 overexpression was induced, and samples were collected 48 hours after overexpression. On the day of collection, MLN was added to the 15 cm dish of the control group at 1 μM for 6 hours; In the S32, 1.6 mL of lysis buffer was added to each dish, and the cells were scraped and collected into a tube, rotated on a shaker at 4°C for 10 minutes, and then centrifuged at 1200 rpm in a 4°C low-temperature centrifuge for 15 minutes.

9. A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry according to claim 1, characterized in that In S33, the supernatants of the two tubes were combined into one sample and filtered using a 0.45 µm filter. The sample was then filtered into a tube containing 60 µL of FLAG Beads and rotated on a shaker at 4 °C for 3 h. In the above S34, the sample and FLAG Beads mixture was transferred to a tube and washed with MCLB buffer three times, 15 minutes each time; then eluted with 3×FLAG peptide, 200 μL each time, for a total of 2 times, and the eluates were combined into an EP tube.

10. A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry according to claim 1, characterized in that In S35, the sample was added with TCEP at a final concentration of 10 mM and reduced at 37°C for 1 hour. The sample was then alkylated with iodoacetamide at a final concentration of 15 mM. The sample was precipitated with TCA / acetonitrile and resuspended in 50 µL of 0.2 M EPPS pH 8.0 buffer. 1 µL of LysC was added to the resuspension and incubated at room temperature overnight. To the S36, 1 µL of trypsin was added and digested at 37°C for 6 hours. Then, 15 µL of acetonitrile (ACN) was added and mixed, and the mixture was reacted at room temperature for 10 minutes. 4 µL of TMT was added and the mixture was reacted at room temperature for 1.5 hours.

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