A proteomic method for detecting post-translational modifications of radiopharmaceutical-regulated proteins

Abstract

The present invention discloses a proteomics method for detecting post-translational modifications of radiopharmaceutical regulatory proteins, which relates to the field of radiopharmaceuticals, and in particular, uses multi-faceted proteomics technology to study the key regulatory proteins of radiopharmaceuticals in tumor treatment. The method can evaluate the changes in proteins regulated by radiopharmaceuticals and their post-translational modifications (PTMs). Radionuclide drugs kill tumor cells through the alpha particles and beta particles released after the decay of their unstable nuclei. The currently recognized mechanism of action of radionuclide drugs is to induce tumor cell DNA damage, thereby leading to tumor cell death, but the specific changes at the specific protein and PTM levels behind this mechanism are still unclear. Traditional research methods cannot comprehensively evaluate the overall effect of radiopharmaceuticals at the protein level. Therefore, it is necessary to develop a research method that can deeply explore the key regulatory proteins of radionuclide drugs for treating tumors from multiple angles at the molecular level.

Description

Technical Field

[0001] The present invention relates to the field of radiopharmaceuticals, and in particular to a proteomics method for detecting post-translational modification of radiopharmaceutical-regulated proteins. Background Art

[0002] Radionuclide drugs are widely used in clinical medicine for the diagnosis and treatment of diseases, especially in oncology. Radionuclides can not only locate and track tumor cells and scan and image tumors through nuclear medicine imaging diagnosis, but also use the high-energy rays released by radionuclides to irradiate tumor cells, or use radionuclide drug conjugates (RDCs), that is, radionuclides are coupled with ligands such as monoclonal antibodies, peptides or small molecules, and the targeting effect of ligands is used to accurately deliver radionuclides to the tumor, while reducing the radiation to normal tissues while killing the tumor at high doses, thereby reducing side effects. At present, the recognized drug effect of radionuclides is to destroy tumor cell DNA through high-energy rays, resulting in double-strand or single-strand breaks in DNA, thereby causing tumor cell death. Traditional research methods for the effects of radiopharmaceuticals are usually targeted at a specific molecular target, such as enzyme-linked immunosorbent assay, immunoblotting and flow cytometry. Although this molecular-specific research approach can generate important biological insights, the effects of radionuclide drugs on tumor cells are often extensive and complex, and only analyzing a certain pathway or molecule cannot achieve a comprehensive analysis of the molecular spectrum regulated by radionuclide drugs. Therefore, detecting the key regulatory proteins of radionuclide drugs at different omics levels and conducting a comprehensive and systematic analysis of the effects of radionuclide drugs at the overall protein level will provide comprehensive molecular guidance for the clinical diagnosis and treatment of radionuclide drugs in the future.

[0003] In recent years, proteomics and post-translational modification (PTMs) proteomics have been increasingly used in clinical tumor diagnosis and treatment. Proteins are the direct executors of many biological processes and the targets of most drugs. The diversity of protein functions is mainly regulated by their PTMs, so the study of PTMs is of great significance for revealing the key protein changes regulated by radionuclide drugs from different angles.

[0004] However, proteomics techniques are rarely used in the study of radionuclide drugs. Therefore, it is urgent to develop a technology to achieve a panoramic analysis of the changes in proteins and post-translational modifications affected by radionuclides. By developing such an experimental process and data processing method, it will help to deeply and comprehensively detect the changes in the proteome and post-translational modification group in tumor cells and tumor microenvironment after radionuclide administration, and can efficiently explore the key regulatory proteins and their modifications related to the action of radionuclides, providing new guidance for radionuclide resistance and clinical combination therapy. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a proteomics method for detecting post-translational modification of radiopharmaceutical regulated proteins.

[0006] The technical solution of the present invention is as follows:

[0007] A proteomic method for detecting post-translational modification of a radiopharmaceutical regulated protein comprises the following steps:

[0008] S1, biological sample protein extraction process;

[0009] A biological sample treated with a radioactive drug is selected, and after being broken and protein extracted in a protein denaturing solution, a reduction alkylation reaction is performed, and then a protease is added to enzymatically hydrolyze the sample to obtain an enzymatically hydrolyzed polypeptide sample of the biological sample;

[0010] S2, enzymatic hydrolysis and enrichment of glycopeptides and phosphorylated peptides;

[0011] Glycopeptide enrichment materials and phosphorylated peptide enrichment materials were used to selectively enrich the enzymatic peptide samples to obtain enriched glycopeptide samples and phosphorylated peptide samples. The load effluent generated during the glycopeptide or phosphorylated peptide enrichment process was used as a protein sample. The glycopeptide samples, phosphorylated samples and protein samples obtained above were analyzed and identified using liquid chromatography-mass spectrometry tandem instruments.

[0012] As a preferred embodiment of the present invention, in step S1, the radioactive drug includes radiotherapy or radionuclide-coupled drugs, such as nuclide-coupled monoclonal antibodies, polypeptides or small molecules.

[0013] As a preferred embodiment of the present invention, in step S1, the protein denaturing solution contains one or more combinations of 0-8M urea, 0-8M guanidine hydrochloride, and 0%-10wt% SDS;

[0014] The crushing method includes at least one of ultrasonic crushing and grinding crushing;

[0015] The reductive alkylation reaction includes a reduction process and an alkylation process. The reduction process uses a 5-100 mM ammonium bicarbonate solution containing 5-50 mM dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride to react for 5-120 minutes under heating; the alkylation process specifically uses a 5-100 mM ammonium bicarbonate solution containing 5-50 mM iodoacetamide or chloroacetamide to react for 5-60 minutes at 25-30° C. in the dark.

[0016] As a preferred embodiment of the present invention, in step S1, the protease is one or more combinations of trypsin, chymotrypsin, lysyl endonuclease, thermolysin, proteinase K, and glutamyl endonuclease;

[0017] The mass ratio of the protease to the sample is 1:1-1:50; and the enzymolysis time is 30 minutes to 24 hours.

[0018] As a preferred embodiment of the present invention, in step S2, the enrichment process of glycopeptides and phosphorylated peptides adopts solid phase extraction mode (SPE) or dispersed solid phase extraction mode (dSPE);

[0019] The glycopeptide enrichment material is a hydrophilic chromatography material: at least one of ZIC-HILIC, TiO2, IMAC, SAX, and amino silica gel; or a laboratory-made glycopeptide enrichment material, such as one or a combination of Click-Mal, TE-Cys, and HBS; the mass ratio of the amount thereof to the enzymatic peptide sample is 100:1-1000:1;

[0020] The phosphorylated peptide enrichment material includes IMAC, TiO2, and ZrO2, and the mass ratio of the phosphorylated peptide enrichment material to the enzymatically hydrolyzed peptide sample is 100:1-1000:1.

[0021] The present invention also discloses a method for analyzing key regulatory proteins in the action of radiopharmaceuticals based on multi-faceted proteomics, comprising the following steps:

[0022] (1) using liquid chromatography-mass spectrometry to detect and analyze any of the above-mentioned glycopeptide samples and phosphorylated peptide samples to obtain mass spectrometry data;

[0023] (2) Database retrieval of mass spectrometry data to conduct qualitative and quantitative analysis of glycopeptides and phosphorylated peptides;

[0024] (3) The quantitative values ​​of protein and post-translationally modified peptide abundance between the nuclide administration group and the control group obtained in step (2) were subjected to differential analysis, differential protein Gene Ontology functional enrichment analysis, differential protein KEGG pathway enrichment analysis and protein interaction network analysis.

[0025] As a preferred embodiment of the present invention, in step (1), the detection and analysis conditions of liquid chromatography-mass spectrometry are as follows: Specifically (1) using any one of the Orbitrap series mass spectrometers of Thermo Fisher and the TIMS-TOF mass spectrometer of Bruker and a matching liquid chromatography system instrument;

[0026] The liquid chromatography analysis column and pre-column adopt conventional C18 capillary column, the capillary filling length is 10-50cm, and the C18 filler particles used are 1.7-5μm; the liquid phase mobile phase adopted is a mixed solvent of organic solvent, deionized water, and organic acid, the organic acid is one of formic acid, acetic acid or trifluoroacetic acid, and the volume proportion of organic acid is 0.1%-30%; the volume proportion of organic solvent is 1-80%;

[0027] The chromatographic separation mode adopted is the reverse phase chromatographic separation mode; the acquisition mode of glycoproteomics data is the data dependent acquisition mode (DDA), and the acquisition mode of phosphoproteomics data is any one of the data dependent acquisition mode (DDA) and the data independent acquisition mode (DIA).

[0028] As a preferred embodiment of the present invention, the glycoproteomics data retrieval software uses one or several combinations of Byonic software from Protein Metrics or open source software such as pGlyco software; the phosphoproteomics data retrieval software uses one or several combinations of Maxquant, PD software from Thermo Fisher, Spectronaut software from Biognosys or open source software such as DIA-NN; aminomethylation of cysteine ​​residues is a fixed modification, and oxidation of methionine is a variable modification; the false positive rates at both the parent ion and peptide levels are set to 1%;

[0029] The differential analysis includes filtering and filling of missing values, log2 transformation, and significance test of differences, wherein differentially expressed proteins or peptides are defined as |log2 fold change|>1.5 and p≤0.05;

[0030] The Gene Ontology functional enrichment and KEGG pathway enrichment analysis of differential proteins is to use the DAVID database to perform GO functional annotation and KEGG pathway analysis on the obtained differential protein set;

[0031] The protein interaction network analysis is to use the String database and Cytoscape software to perform interaction network analysis on the obtained differential proteins.

[0032] The beneficial effects of the present invention are: the method of the present invention can evaluate the changes of proteins and their post-translational modifications (PTMs) regulated by radiopharmaceuticals, obtain a more comprehensive protein and post-translational modification data set, and mine its key regulatory proteins from multiple post-translational modification levels. It solves the problem that traditional research methods cannot comprehensively evaluate the overall effects of radiopharmaceuticals at the protein level. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention is based on proteomics and post-translational modification omics 225 Schematic diagram of the analysis process of key regulatory proteins of Ac.

[0034] Figure 2 The detected 225 Volcano plot of site-specific glycans that underwent significant differential changes in tumor tissues after Ac treatment.

[0035] Figure 3 The detected 225 Pathways involving proteins belonging to site-specific glycans that undergo significant differences in tumor tissues after Ac treatment.

[0036] Figure 4 The detected 225 Protein interaction network related to immune regulation among key glycoproteins regulated by Ac.

[0037] Figure 5 The detected 225 After Ac treatment, the protein abundance of the key regulatory protein Igtam was upregulated in tumor tissues, the expression of the sugar chain H8N2 on the glycosylation site N734 of Igtam was upregulated, and the expression of the sugar chain H9N2 on the glycosylation site N907 of Igtam was downregulated.

[0038] Figure 6 The detected 225 Volcano plot of phosphorylation sites that underwent significant differential changes in tumor tissues after Ac treatment.

[0039] Figure 7 The detected 225 The kinases corresponding to the phosphorylation sites that underwent significant differences in tumor tissues after Ac treatment.

[0040] Figure 8 The detected 225 Protein interaction network related to cell cycle regulation among key phosphorylated proteins regulated by Ac.

[0041] Fig. 9It is a common regulatory pathway in proteomics, phosphoproteomics and glycoproteomics in the present invention. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] The present invention provides a proteomics sample processing and data analysis technology for studying key proteins regulated by radiopharmaceuticals. The advantage of the present invention is that, compared with the traditional target-specific drug action analysis method, the present invention can obtain a more comprehensive protein and post-translational modification data set and mine its key regulatory proteins from multiple post-translational modification levels.

[0044] The technical solution adopted by the present invention is as follows:

[0045] In a first aspect, the present invention provides a sample pretreatment method for detecting key regulatory proteins of radioactive drugs based on multifaceted proteomics, wherein the sample pretreatment method includes a protein extraction process of a biological sample, an enzymatic hydrolysis process, and an enrichment process of glycopeptides and phosphorylated peptides.

[0046] The biological sample is a biological sample tissue treated with a radioactive drug, and the radioactive drug includes a traditional radiotherapy or a radionuclide-coupled drug, such as a nuclide-coupled monoclonal antibody, a peptide or a small molecule, etc.; the protein denaturing solution contains one or more combinations of urea with a concentration of 0-8M, guanidine hydrochloride with a concentration of 0-8M, and SDS with a concentration of 0%-10%; the protein crushing method includes an ultrasonic crushing method and a grinding crushing method; after the protein extraction process is completed, the obtained protein solution is centrifuged at a centrifugal speed of 10000-14000rpm and a centrifugal time of 10-30min, and the obtained supernatant is the protein solution to be tested, and the concentration of the protein sample is determined by a BCA protein assay.

[0047] Wherein, the proteolysis process of the biological sample is to perform reduction alkylation and enzymolysis reaction on the obtained protein solution to be tested. The protein reduction process includes using a 5-100mM ammonium bicarbonate solution containing dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine hydrochloride (TCEP) at a concentration of 5-50mM, reacting for 5-120 minutes under heating conditions, and the temperature of the heating treatment is 37-100°C; the protein alkylation process includes using a 5-100mM ammonium bicarbonate solution containing iodoacetamide (IAA) or chloroacetamide (CAA) at a concentration of 5-50mM, and reacting for 5-60 minutes at 25°C in the dark. The proteolysis process includes adding enzymes according to a mass ratio of enzyme to protein of 1:1-1:50, the enzyme cutting time is 30min-24h, the types of proteases include one or more combinations of trypsin, chymotrypsin, lysyl endonuclease, thermolysin, proteinase K, and glutamyl protein endonuclease, and the temperature range of the enzymolysis is the active temperature of the selected protease. After the enzyme digestion process is completed, a formic acid or trifluoroacetic acid solution with a volume fraction of 1-10% is used to terminate the enzyme digestion process to form an enzymatic polypeptide sample of the biological sample.

[0048] The enrichment process of glycopeptides and phosphorylated peptides of the enzymatically hydrolyzed polypeptide sample of the biological sample adopts solid phase extraction mode (SPE) or dispersed solid phase extraction mode (dSPE) to continuously enrich glycopeptides and phosphorylated peptides.

[0049] Wherein, the glycopeptide enrichment material is a hydrophilic chromatography material: at least one of ZIC-HILIC, TiO2, IMAC, SAX, and amino silica gel; or a laboratory-made glycopeptide enrichment material, such as one or a combination of Click-Mal, TE-Cys, and HBS; the material dosage is 1-10 mg; the mass ratio of the glycopeptide enrichment material to the polypeptide mixture of the protein sample is 100:1-1000:1. The phosphorylated peptide enrichment material includes commercial IMAC, TiO2, and ZrO2, and the material dosage is 1-10 mg; the mass ratio of the phosphorylated peptide enrichment material to the enzymatic polypeptide sample of the starting biological sample is 100:1-1000:1.

[0050] The second aspect is a data processing and analysis process for key proteins regulated by radioactive drugs based on multi-level proteomics detection. The data processing method includes a liquid phase-tandem mass spectrometry analysis process, database retrieval using glycoproteomics data retrieval software and phosphorylation proteomics data retrieval software, identification and quantitative analysis of proteins and post-translationally modified peptides, and bioinformatics analysis.

[0051] Among them, the mass spectrometer in the liquid phase-tandem mass spectrometry detection and analysis process is any one of the Orbitrap series mass spectrometers of Thermo Fisher and the TIMS-TOF mass spectrometer of Bruker, and the matching liquid chromatography system instrument; the liquid chromatography analysis column and pre-column adopt conventional C18 capillary column, the capillary filling length is 10-50cm, and the C18 filler particles used are 1.7-5mm. The liquid phase mobile phase used is a mixed solvent of organic solvent, deionized water, and organic acid, the organic acid is one of formic acid, acetic acid or trifluoroacetic acid, and the volume proportion of organic acid is 0.1%-30%; the volume proportion of organic solvent is 1-80%; the chromatographic separation mode used is the reverse phase chromatographic separation mode; the acquisition mode of glycoproteomics and phosphorylation proteomics data can be any one of data-dependent acquisition mode (DDA) or data-independent acquisition mode (DIA).

[0052] Among them, the database retrieval analysis process includes the selection of retrieval software and data screening. The glycoproteomics data retrieval software uses one or several combinations of Byonic software from Protein Metrics or open source software such as pGlyco software; the phosphorylation proteomics data retrieval software uses one or several combinations of Maxquant, PD software from Thermo Fisher, Spectronaut software from Biognosys, or open source software such as DIA-NN; aminomethylation of cysteine ​​residues is a fixed modification, and oxidation of methionine is a variable modification; the false positive rates at the parent ion and peptide levels are set to 1%; the difference analysis includes filtering and filling of missing values, log2 transformation, and significance test of differences, wherein differentially expressed proteins or peptides are defined as |log2 fold change|>1.5 and p≤0.05. The differential protein Gene Ontology functional enrichment and KEGG pathway enrichment analysis uses the DAVID database to perform GO functional annotation and KEGG pathway analysis on the obtained differential protein set. The protein interaction network analysis uses the sting database and Cytoscape software to perform interaction network analysis on the obtained differential proteins.

[0053] The technical solution of the present invention is further described below with reference to specific embodiments.

[0054] SDS: sodium dodecylbenzene sulfonate;

[0055] Example 1

[0056] Select radionuclides 225The tumor tissues of mice after Ac administration and the tumor tissues treated with blank control were processed in parallel, and were placed in 50mM ammonium bicarbonate solution (containing 8M urea) for grinding and protein extraction. The resulting protein solution was centrifuged at 14000rpm for 10min, and the concentration of the supernatant was determined by BCA protein assay. A 50mM ammonium bicarbonate solution containing 5mM dithiothreitol (DTT) was added to the resulting protein solution, and reacted at 95°C for 10 minutes; then a 50mM ammonium bicarbonate solution containing 10mM iodoacetamide (IAA) was added, and reacted at 25°C in the dark for 30 minutes; then trypsin was added, the mass ratio of the enzyme to the protein sample was 1:50, and the enzymolysis was performed at 37°C for 16h. After the enzymolysis was completed, the enzymolysis was terminated with 5% formic acid by volume, and the resulting solution was the proteolytic polypeptide sample. The polypeptide sample was spin-dried, desalted with C18, and then redissolved in an aqueous solution containing 80% by volume ACN and 1% by volume TFA.

[0057] The solid phase extraction mode was used for glycopeptide enrichment. The material used was commercial ZIC-HILIC. 3 mg of ZIC-HILIC material was loaded into an Eppendorf gun tip to make a micro SPE solid phase extraction column for glycopeptide enrichment. The obtained glycopeptide eluate was spin-dried to obtain the glycopeptide sample.

[0058] Solid phase extraction mode was used for phosphorylated peptide enrichment. The material used was commercial IMAC material. 2.5 mg of IMAC material was loaded into an Eppendorf gun tip to make a micro SPE solid phase extraction column for phosphorylated peptide enrichment. The phosphorylated peptide eluate obtained was spin-dried to obtain the phosphorylated peptide sample.

[0059] Easy 1200 liquid phase and Orbitrap 480 mass spectrometer were used, equipped with a capillary column with a length of 20 cm and an outer diameter of 360 mm and filled with C18 reverse phase chromatographic filler with a particle size of 1.9 mm as the analytical column, and equipped with a capillary column with a length of 2 cm and an outer diameter of 360 mm and filled with C18 reverse phase chromatographic filler with a particle size of 1.9 mm as the pre-column. The liquid mobile phase A phase was 0.1% (volume fraction) formic acid, and the phase B was 0.1% (volume fraction) formic acid / 80% acetonitrile for reverse phase chromatography separation; the mass spectrometry data acquisition mode for glycoproteomics data was the data dependent acquisition mode (DDA), and the data acquisition mode for phosphoproteomics was the data independent acquisition mode (DIA). The commercialized Byonic software from Protein Metrics was used for glycopeptide mass spectrometry data retrieval, and the Spectronaut software from Biognosys was used for phosphoproteomics data retrieval. The database used was the UniProt-Mouse database, with aminomethylation of cysteine ​​residues as a fixed modification and oxidation of methionine as a variable modification. The false positive rates at both the parent ion and peptide levels were set to 1%.

[0060] The obtained glycoproteomics and phosphorylation proteomics search database data were filtered according to the data of at least one group of samples in the control group and the drug group, and the differentially expressed proteins or peptides were defined as |log2 fold change|>1.5 and p≤0.05. The obtained differential proteins were used to perform GO functional annotation and KEGG pathway analysis on the obtained differential protein set using the DAVID database. The differential proteins were also analyzed for interaction networks using the sting database and Cytoscape software.

[0061] The results are as follows: Figure 2 As shown in Figure 2, by analyzing the differential expression of site-specific sugar chains, a total of 536 significantly changed site-specific sugar chains were identified in this example, of which 156 were upregulated and 380 were downregulated. Subsequently, the GO enrichment analysis of the glycoproteins to which the above-mentioned differentially expressed site-specific sugar chains belonged was performed to gain a deeper understanding of their involvement in cellular functions and processes. The results showed that these glycoproteins were mainly classified as immune system regulation (see Figure 3 ). String protein interaction network analysis was performed on these glycoproteins involved in immune regulation (see Figure 4 ), and Itgam showed the highest degree of interaction, indicating that it may be 225Ac plays an important role in the anti-tumor effect. Therefore, we continued to analyze the glycosylation level and protein abundance changes of Itgam and found that the glycosylation of Itgam at the N734 site was upregulated, while the glycosylation at N907 was significantly downregulated, while the overall protein abundance of Itgam was upregulated (see Figure 5 ), this result suggests that Itgam may be 225 Ac is a potential key regulatory protein.

[0062] In the phosphoproteomic analysis, a total of 795 upregulated phosphorylation sites and 780 downregulated phosphorylation sites were detected (see Figure 6 ). Kinase substrate enrichment analysis revealed changes in specific kinase activity corresponding to differential phosphorylation sites, including MAPK9, CSNK2A1, CDK1, CDK2, and CHEK1, which are related to DNA damage, cell cycle arrest, and DNA repair (see Figure 7 ). The cellular pathways involved in the phosphoproteins with differentially expressed phosphorylation sites were subsequently analyzed, revealing cell cycle-related 225 The interaction network of regulatory proteins of Ac (see Figure 8 ).

[0063] Comprehensive pathway analysis of proteomics, glycoproteomics, and phosphoproteomics data revealed that regulation of the hemostatic system was the most significant pathway shared by the proteome, phosphoproteome, and glycoproteome. Pathways significantly enriched only in the proteome included aerobic respiration, monocarboxylic acid metabolism, acyl-CoA metabolism, carbon metabolism, and mitochondrial protein degradation. Pathways primarily regulated by glycosylation included protein processing in the extracellular matrix organization, lysosomes, and endoplasmic reticulum. Pathways specifically regulated by phosphorylation included chromatin conformation, mRNA metabolism, and DNA damage, among others. Fig. 9 ).

[0064] In summary, Example 1 of the present invention discloses 225 Ac induces changes in multiple signaling pathways, including mitochondrial energy metabolism, immune regulation, hemostasis pathway regulation, and DNA damage and repair, and identifies the immune-related molecular target Igtam and a series of key phosphorylated protein networks related to the cell cycle.

[0065] The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. A proteomics method for detecting post-translational modifications of radiopharmaceutical regulated proteins, characterized in that: The following steps are involved: S1, biological sample protein extraction process; A biological sample treated with a radioactive drug is selected, and after being broken and protein extracted in a protein denaturing solution, a reduction alkylation reaction is performed, and then a protease is added to enzymatically hydrolyze the sample to obtain an enzymatically hydrolyzed polypeptide sample of the biological sample; S2, enzymatic hydrolysis and enrichment of glycopeptides and phosphorylated peptides; Glycopeptide enrichment materials and phosphorylated peptide enrichment materials were used to selectively enrich the enzymatic peptide samples to obtain enriched glycopeptide samples and phosphorylated peptide samples. The load effluent generated during the glycopeptide or phosphorylated peptide enrichment process was used as a protein sample. The glycopeptide samples, phosphorylated samples and protein samples obtained above were analyzed and identified using liquid chromatography-mass spectrometry tandem instruments.

2. A proteomics method for detecting post-translational modification of radiopharmaceutical regulated proteins according to claim 1, characterized in that: In step S1, the radioactive drug includes radiotherapy or radionuclide-coupled drug.

3. A proteomics method for detecting post-translational modification of radiopharmaceutical regulatory proteins according to claim 1, characterized in that: In step S1, the protein denaturing solution contains one or more combinations of 0-8M urea, 0-8M guanidine hydrochloride, and 0%-10wt% SDS; The crushing method includes at least one of ultrasonic crushing and grinding crushing; The reductive alkylation reaction includes a reduction process and an alkylation process. The reduction process uses a 5-100 mM ammonium bicarbonate solution containing 5-50 mM dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride to react for 5-120 minutes under heating; the alkylation process specifically uses a 5-100 mM ammonium bicarbonate solution containing 5-50 mM iodoacetamide or chloroacetamide to react for 5-60 minutes at 25-30° C. in the dark.

4. A proteomics method for detecting post-translational modification of radiopharmaceutical regulatory proteins according to claim 1, characterized in that: In step S1, the protease is one or more combinations of trypsin, chymotrypsin, lysyl endonuclease, thermolysin, proteinase K, and glutamyl endonuclease; The mass ratio of the protease to the sample is 1:1-1:50; and the enzymolysis time is 30 minutes to 24 hours.

5. A proteomics method for detecting post-translational modification of radiopharmaceutical regulated proteins according to claim 1, characterized in that: In step S2, the enrichment process of glycopeptides and phosphorylated peptides adopts a solid phase extraction mode or a dispersed solid phase extraction mode; The glycopeptide enrichment material is a hydrophilic chromatography material, and the mass ratio of the glycopeptide enrichment material to the enzymatic peptide sample is 100:1-1000:1; The phosphorylated peptide enrichment material includes IMAC, TiO2, and ZrO2, and the mass ratio of the phosphorylated peptide enrichment material to the enzymatically hydrolyzed peptide sample is 100:1-1000:

1.

6. A method for identifying key regulatory proteins in the action of radiopharmaceuticals based on multifaceted proteomics analysis, characterized in that: The following steps are involved: (1) using liquid chromatography-mass spectrometry to detect and analyze the glycopeptide sample and phosphorylated peptide sample according to any one of claims 1 to 5 to obtain mass spectrometry data; (2) Database retrieval of mass spectrometry data to conduct qualitative and quantitative analysis of glycopeptides and phosphorylated peptides; (3) The quantitative values ​​of protein and post-translationally modified peptide abundance between the nuclide administration group and the control group obtained in step (2) were subjected to differential analysis, differential protein Gene Ontology functional enrichment analysis, differential protein KEGG pathway enrichment analysis and protein interaction network analysis.

7. A proteomics method for detecting post-translational modification of radiopharmaceutical regulatory proteins according to claim 6, characterized in that: In step (1), the detection and analysis conditions of liquid chromatography-mass spectrometry are as follows: The liquid chromatography analysis column and pre-column adopt conventional C18 capillary column, the capillary filling length is 10-50cm, and the C18 filler particles used are 1.7-5μm; the liquid phase mobile phase adopted is a mixed solvent of organic solvent, deionized water, and organic acid, the organic acid is at least one of formic acid, acetic acid or trifluoroacetic acid, and the volume proportion of organic acid is 0.1%-30%; the volume proportion of organic solvent is 1-80%; The chromatographic separation mode adopted is the reverse phase chromatographic separation mode; the acquisition mode of glycoproteomics data is the data-dependent acquisition mode, and the acquisition mode of phosphoproteomics data is any one of the data-dependent acquisition mode and the data-independent acquisition mode.

8. A proteomics method for detecting post-translational modification of radiopharmaceutical regulated proteins according to claim 6, characterized in that: The differential analysis includes filtering and filling of missing values, log2 transformation, and significance test of differences, wherein differentially expressed proteins or peptides are defined as |log2fold change|>1.5 and p≤0.05; The Gene Ontology functional enrichment and KEGG pathway enrichment analysis of differential proteins is to use the DAVID database to perform GO functional annotation and KEGG pathway analysis on the obtained differential protein set; The protein interaction network analysis is to use the String database and Cytoscape software to perform interaction network analysis on the obtained differential proteins.

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