PROTAC research and development method for identifying CRL compound based on ARIH1 mass spectrum

Abstract

The invention discloses a PROTAC research and development method for identifying a CRL compound based on ARIH1 mass spectrum, and relates to the technical field of biology. Comprising the following steps: S1, constructing a PBTE-C357SARIH1 overexpressed H9 cell line; s2, inducing the H9 stem cell line to differentiate from ESC to mesoderm and endoderm; s3, carrying out an over-expression ARIH1 immunoprecipitation experiment; the method comprises the following steps: S31, respectively inoculating 12 15cm cell vessels with H9 cells, respectively adding 20mL of an RPMI culture medium, simultaneously adding Doxycycline to induce FLAG-ARIH1 overexpression, and preparing to collect a sample after overexpression. On the basis of the characteristic that ARIH1 serves as CO-E3 ligase and forms a stable compound with CRL subjected to quasi-activation, a CRL substrate receptor in an activated state is accurately identified by utilizing a TMT-IP / MS technology.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a PROTAC research and development method based on ARIH1 mass spectrometry identification of CRL complexes. Background Art

[0002] The Cullin-RING ubiquitin ligase (CRL) family is the largest E3 ubiquitin ligase family in the ubiquitin-proteasome system, regulating protein degradation by recognizing specific substrate receptors. The CRL family plays a key role in a variety of 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 in their activated state has always been a research difficulty.

[0003] TMT-IP / MS (Tandem Mass Tag Immunoprecipitation Mass Spectrometry) is an advanced method that combines TMT peptide labeling technology and immunoprecipitation mass spectrometry technology. It is widely used in protein interaction research, especially in the development of PROTAC (protein degradation targeting chimera). TMT (Tandem Mass Tag) technology is an in vitro peptide labeling technology developed by ThermoScientific, USA. This technology uses isotope tags to achieve quantitative analysis of peptides in mass spectrometry analysis by specifically labeling the amino group of the peptide. TMT technology has the characteristics of high sensitivity, high throughput and high accuracy. It can perform quantitative analysis on multiple samples at the same time and is suitable for complex proteomics research. IP / MS (Immunoprecipitation-Mass Spectrometry) is a protein interaction research method based on immunoprecipitation and mass spectrometry analysis. Its principle is to capture the target protein and its interacting proteins with specific antibodies, 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. Quantitative analysis of immunoprecipitated peptides by TMT labeling technology can more accurately identify and quantify protein interactions. With its high specificity, high sensitivity and quantitative analysis capabilities, TMT-IP / MS technology has important application value in PROTAC development and 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 mechanism 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 its molecular mechanism for substrate recognition. These studies provide an important basis for understanding the functions of the CRL family, but they mainly focus on structural analysis and mechanism research, and only focus on a certain substrate of CRL, and have not yet involved the use of TMT-IP / MS technology to systematically identify multiple substrate receptors in an activated state.

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

[0006] ① Drugability issues caused by large molecular weight: Traditional small molecule drugs basically comply with the drugability principle - Lipinski's "five rules of drug-like drugs", but PROTAC molecules often have large molecular weights. The molecular weights of PROTACs reported so far are 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 drugability.

[0007] ②The number of available E3 ubiquitin ligases is limited: As one of the components of PROTAC, E3 ubiquitin ligase plays a vital role. Although studies have found that more than 600 E3 ligases can play a role in human cells, most E3 ligases have limited tissue distribution. Even the most commonly used E3 ligases, CEBN, MDM2, and XIAP, still have a small number of tissues that have not been covered. For example, MDM2 ligase is present in 99% of the test samples, while CRBN is only expressed in 76% of the test samples. Therefore, there is still a lot of room for the development of E3 ligases with wide tissue distribution and ligands with good drug properties. Among the more than 600 E3 ligases, only 12 E3 ligases have been used in PROTACs, two of which have entered the clinical trial stage, and the other 10 are in the preclinical exploration stage, currently mainly targeting 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 will inevitably occur, which brings difficulties to the rational design of in vivo doses.

[0009] The CRL (Cullin RING ubiquitin ligase) family is the largest E3 ubiquitin ligase superfamily, containing more than 200 family members, regulating the ubiquitination degradation of about 20% of proteins in cells. As the most important E3 superfamily, the biochemical activity of CRL has a precise regulatory system: CRL is a modular complex enzyme, and its Cullin protein can be used as a scaffold to assemble hundreds of different substrate receptors, thereby ubiquitinating specific substrate proteins. The substrate proteins of CRL include key proteins such as Cyclin D, HIF1α, AKT, Nrf2, PCNA, PD-1, PD-L1, etc., and therefore play an important regulatory role in biological processes such as cell cycle, signal transduction, DNA damage repair, and immune checkpoints. Abnormal expression or activity of multiple CRL family members is involved in the occurrence of diseases such as tumors and is an important therapeutic target. In addition, CRL4CRBN and CRL2VHL are also E3 ubiquitin ligases widely used in PROTAC technology. The successful approval of ARV-110 based on CRL4CRBN shows that CRL has great potential and space in this emerging new drug research and development field. Although CRL has important biological functions and plays a key regulatory role in many diseases, compared with kinases that catalyze phosphorylation modifications, the development of small molecule drugs targeting CRL ubiquitin ligases is far behind. One of the main reasons is that research on the regulatory mechanism and function of the CRL family is relatively lacking.

[0010] Since ARIH1 can form a stable complex with Cullin-RING ubiquitin ligase (Cullin-RING ubiquitin ligase) that is activated by pseudotinylation as a CO-E3, the present invention mainly uses TMT-IP / MS (Tandem Mass Tag immunoprecipitation mass spectrometry) to identify CRL substrate receptors in an activated state on ARIH1, and screens and identifies CRL family members with key biological functions. Summary of the invention

[0011] The purpose of the present invention is to solve the shortcomings of the prior art and propose a PROTAC development method based on ARIH1 mass spectrometry identification of CRL complexes.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry, comprising the following steps:

[0014] S1: Construction of PBTE-C357S ARIH1 overexpressing 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 inoculated into 12 15 cm cell dishes, 20 mL RPMI medium was added, and Doxycycline was added to induce FLAG-ARIH1 overexpression. After overexpression, samples were prepared for collection. On the day of collection, MLN treatment was added to the control group;

[0018] S32: Lyse cells using MCLB buffer containing protease inhibitors. 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 using 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 it for 1 hour, then alkylate the sample with iodoacetamide; precipitate with TCA / acetonitrile, then resuspend in buffer, add LysC to the resuspended solution, and incubate at room temperature overnight;

[0022] S36: The next day, trypsin was added and digested for 6 hours, then acetonitrile (ACN) was added and mixed, and reacted at room temperature; TMT was added and reacted again at room temperature;

[0023] S37: The cells differentiated into mesoderm and endoderm were treated in the same way, and then 2 μL was taken from each sample, combined into 100 μL 5% FA / 5% ACN, treated with Stage tip, and subjected to mass spectrometry analysis after drying.

[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 reach a confluence of 70%-90% at the time of transfection;

[0026] S12: Prepare plasmid DNA and transfection reagents, calculate the required amount of PBTE-C357SARIH1 and hypbase plasmids, and mix them in a ratio of 3:1;

[0027] S13: Add 100 μL of serum-free Opti-MEM medium to a sterile centrifuge tube, then add 2 μg of plasmid DNA, 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, 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;

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

[0029] S15: Transfection, wait for the set time and then change the medium, replace it with a complete medium containing serum, and add hygromycin B for screening. When the control cells die by more than 80%, remove the antibiotics in the experimental group and replace it with a 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 wells for WB detection of ARIH1 expression.

[0031] Preferably, in said S14, specifically: placing the culture plate in an incubator at 37° C. and 5% CO 2 for continued culture, and replacing it with a complete culture medium containing serum 6 hours after transfection, 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, and 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 state is waited 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 cells overexpressing ARIH1 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 time the cells have differentiated into the mesoderm, and the subsequent induction medium is prepared and the medium is replaced;

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

[0043] Preferably, in 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 S31, H9 cells are cultured at 2.7×10 7 The cells were inoculated into 12 15 cm cell dishes at a density of 10 cells each, 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 prepared 48 hours after overexpression. On the day of sample collection, MLN was added to the 15 cm dish of the control group, and 1 μM was treated for 6 hours;

[0049] In the S32, 1.6 mL of lysis buffer was added to each dish, cells were scraped and collected into tubes, 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] Preferably: in said S33, the supernatants of the two tubes are combined into one sample, filtered using a 0.45 μm filter, and then filtered into a tube containing 60 μL FLAG Beads, and rotated on a shaker at 4°C for 3 hours;

[0051] In the S34, the sample and FLAG Beads mixture was transferred to a tube and washed with MCLB buffer for 3 times, 15 minutes each time; then 3×FLAG peptide was used for elution, 200 μL each time, for a total of 2 times, and the eluates were combined into an EP tube.

[0052] Preferably: in said S35, TCEP with a final concentration of 10 mM is added to the sample, and the sample is reduced at 37°C for 1 hour, and then iodoacetamide with a final concentration of 15 mM is used to alkylate the sample; TCA / acetonitrile is used for precipitation, and then the sample is resuspended in 50 μL 0.2M EPPS pH 8.0 buffer, and 1 μL LysC is added to the resuspended solution, and the sample is kept at room temperature overnight;

[0053] In the S36, 1 μL of trypsin was added and digested at 37° C. for 6 hours, and then 15 μL of acetonitrile (ACN) was added and mixed, and reacted at room temperature for 10 minutes; 4 μL of TMT was added and reacted at room temperature for 1.5 hours.

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

[0055] 1. Based on the property that ARIH1, as a CO-E3 ligase, forms a stable complex with CRL activated by pseudotype, the present invention uses TMT-IP / MS technology to accurately identify CRL substrate receptors in an activated state.

[0056] 2. The technology of the present invention can be applied to different cell lines, including a variety of tumor cell lines, which is helpful 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 development of new PROTACs.

[0057] 3. The technology of the present invention can quickly identify key CRL substrate receptors and provide clear targets for the design of PROTAC, thereby shortening the cycle from target discovery to drug design; through precise screening and identification, it reduces investment in invalid targets and reduces R&D costs.

[0058] 4. The technology of the present invention is applicable to a variety of cell lines, including different tumor cell lines, and can systematically study the dynamic assembly and functional regulation of the CRL family in different cancers; this provides the possibility for the development of personalized PROTACs for specific tumor types; through application in different tumor cell lines, tumor-specific CRL substrate receptors can be discovered, thereby developing more precise tumor therapeutic 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 in the present invention provide the possibility for the development of PROTACs targeting these "undruggable" targets. Each PROTAC molecule can degrade multiple target protein molecules and has catalytic degradation function, so only a low dose is needed to exert significant efficacy, and the efficacy is long-lasting. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0061] Figure 2 The volcano plot analysis result of the effects of ARIH1 overexpression and MLN4924 treatment on substrate receptor activity in human embryonic stem cells of the present invention is shown;

[0062] Figure 3 It is a volcano plot analysis result diagram of the effects of ARIH1 overexpression and MLN4924 treatment on substrate receptor activity in human mesodermal cells of the present invention;

[0063] Figure 4 It is a volcano plot analysis result diagram of the effects of ARIH1 overexpression and MLN4924 treatment on substrate receptor activity in human endoderm cells of the present invention;

[0064] Figure 5 Schematic diagram of the activity states of multiple substrate receptors in different cell types of the present invention. DETAILED DESCRIPTION

[0065] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0066] Embodiment 1:

[0067] A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry, comprising the following experimental steps:

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

[0069] S11: One day before transfection, plate H9 cells at an appropriate density (about 0.5-2×10 per well). 5 Cells were inoculated into 6-well plates and 2 mL of culture medium without antibiotics was added to ensure that the cell confluence reached 70%-90% during transfection to obtain the best transfection efficiency;

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

[0071] S13: Add 100 μL serum-free Opti-MEM medium (Gibco) to a sterile centrifuge tube, then add 2 μg plasmid DNA (PBTE-C357S ARIH1 and hypbase plasmids are mixed in proportion), gently mix, add 100 μL serum-free Opti-MEM medium to another sterile centrifuge tube, then add 5 μL Lipofectamine 2000 (ThermoScientific), gently mix and let stand at room temperature for 5 minutes, add the DNA dilution to the Lipofectamine 2000 dilution, gently mix, 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 gently shake the culture plate to evenly distribute the complex;

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

[0073] S15: 48 hours after transfection, the medium was changed to complete medium containing serum, and hygromycin B (sangon, 100 μg / ml) was added for screening. When more than 80% of the control cells died, the antibiotics of the experimental group were removed, and the medium was replaced with complete medium containing serum, and the cells were allowed to return to normal.

[0074] S16: After the stably transfected cells can be passaged normally, the expression of ARIH1 is detected by WB in each well (detected by FLAG tag antibody). Compared with the control, an obvious overexpression band (~70kDa) appears in the experimental group, indicating that the stable cell line is successfully constructed.

[0075] S2: Induce H9 stem cell line to differentiate from ESC (Embryonic Stem Cells) to mesendoderm and 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 was used, and the following reagents were added per ml: Glutamax (100×) 10 μL, CHIR99021 (5 μM) 1 μL, 200 ng / mL Activin A 0.2 μL). From this time 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 time the cells have differentiated into mesendoderm, prepare the subsequent induction medium and change the medium (using Advanced RPMI 1640 medium, adding the following reagents per ml: Glutamax (100×) 10 μL, 100 ng / mL Activin A 1 μL);

[0080] S25: On the fifth day, the 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 12 cells) were inoculated into 12 15 cm cell dishes (6 experimental groups and 6 control groups), and 20 mL RPMI medium (Gibco) was added. Doxycycline (0.5 mg / ml) was also added to induce FLAG-ARIH1 overexpression. Samples were prepared 48 h after overexpression. On the day of sample collection, MLN (1 μM) (MLN: a pseudotype inhibitor that can inactivate CRL) was added to the 15 cm dish of the control group for 6 h.

[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, pH adjusted to 7.4 with HCl) containing protease inhibitors. Before lysis, add protease inhibitors (MCE) to the lysis buffer, and 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, and then centrifuge at 12,000 rpm in a 4°C low-temperature centrifuge for 15 min.

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

[0085] S34: Transfer the sample and FLAG Beads mixture to a 15 mL tube and wash with MCLB buffer three times, 15 minutes each time. Then use 3×FLAG peptide (Cytiva) for elution, 200 μL each time, for a total of 2 times, and combine the eluates into a 1.5 ml EP tube;

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

[0087] S36: The next day, 1 μL trypsin (Gibco) was added and digested at 37°C for 6 hours, and then 15 μL acetonitrile (ACN) was added and mixed, and reacted at room temperature for 10 minutes. 4 μL TMT (Thermo Scientific) was added and reacted at room temperature for 1.5 hours;

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

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

[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). The dark squares indicate that the substrate receptor is not detected (ND) or inactive (Inactive) in a specific cell type, while the light squares indicate that the substrate receptor is active (Active) in a specific cell type, which 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 implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A PROTAC development method for identifying CRL complexes based on ARIH1 mass spectrometry, 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 inoculated into 12 15 cm cell dishes, 20 mL RPMI medium was added, and Doxycycline was added to induce FLAG-ARIH1 overexpression. After overexpression, samples were prepared for collection. On the day of collection, MLN treatment 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 cells and collect them into tubes, rotate on a shaker, and then centrifuge using a low-temperature 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 it for 1 hour, then alkylate the sample with iodoacetamide; precipitate with TCA / acetonitrile, then resuspend in buffer, add LysC to the resuspended solution, and incubate at room temperature overnight; S36: The next day, trypsin was added and digested for 6 hours, then acetonitrile (ACN) was added and mixed, and reacted at room temperature; TMT was added and reacted again at room temperature; S37: The cells differentiated into mesoderm and endoderm were treated in the same way, and then 2 μL was taken from each sample, combined into 100 μL 5% FA / 5% ACN, treated with Stage tip, and subjected to mass spectrometry analysis after drying.

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 reach a confluence of 70%-90% at the time of transfection; S12: Prepare plasmid DNA and transfection reagents, calculate the required amount of PBTE-C357SARIH1 and hypbase plasmids, and mix them in a ratio of 3:1; S13: Add 100 μL of serum-free Opti-MEM medium to a sterile centrifuge tube, then add 2 μg of plasmid DNA, mix well, add 100 μL of serum-free Opti-MEM medium to another sterile centrifuge tube, then add 5 μL of Lipofectamine2000, mix well and let stand at room temperature for 5 minutes, add the DNA dilution to the Lipofectamine 2000 dilution, mix well, let stand at room temperature for 20 minutes to form a DNA-Lipofectamine 2000 complex, slowly add the prepared DNA-Lipofectamine2000 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 an incubator and continue culturing. After transfection, replace the culture medium with complete medium containing serum and continue culturing. S15: Transfection, wait for the set time and then change the medium, replace it with a complete medium containing serum, and add hygromycin B for screening. When the control cells die by more than 80%, remove the antibiotics in the experimental group and replace it with a complete medium containing serum, and wait for the state to return to normal; S16: After the stably transfected cells can be passaged normally, separate wells for WB detection of ARIH1 expression.

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, and after 6 hours of transfection, the culture medium is replaced with a complete medium containing serum, and the 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 the 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 state is waited 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 cells overexpressing ARIH1 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: Glutamax (100×): 10 μL; CHIR99021 (5 μM): 1 μL; 200ng / 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 time the cells have differentiated into the mesoderm, and the subsequent induction medium is prepared and the medium is replaced; 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 S21, for a 6-well plate, 1 × 10 5 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: Glutamax (100×): 10 μL; 100ng / 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 S31, H9 cells were cultured at 2.7×10 7 The cells were inoculated into 12 15 cm cell dishes at a density of 10 cells each, 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 prepared 48 hours after overexpression. On the day of sample collection, MLN was added to the 15 cm dish of the control group, and 1 μM was treated for 6 hours; In the S32, 1.6 mL of lysis buffer was added to each dish, cells were scraped and collected into tubes, 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, then filtered into a tube containing 60 μL of FLAG Beads and rotated on a shaker at 4°C for 3 hours; In the S34, the sample and FLAG Beads mixture was transferred to a tube and washed with MCLB buffer for 3 times, 15 minutes each time; then 3×FLAG peptide was used for elution, 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 the S35, TCEP with a final concentration of 10 mM was added to the sample, and the sample was reduced at 37°C for 1 hour, and then alkylated with iodoacetamide with a final concentration of 15 mM; the sample was precipitated with TCA / acetonitrile, and then resuspended in 50 μL 0.2M EPPS pH8.0 buffer, and 1 μL LysC was added to the resuspended solution, and incubated at room temperature overnight; In the S36, 1 μL of trypsin was added and digested at 37° C. for 6 hours, and then 15 μL of acetonitrile (ACN) was added and mixed, and reacted at room temperature for 10 minutes; 4 μL of TMT was added and reacted at room temperature for 1.5 hours.

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