Preparation method of tumor cell-specific modified polypeptide / protein, antibody and tumor diagnosis and treatment method

Abstract

The present application discloses a method for preparing modified polypeptides / proteins specific to tumor cells, antibodies, and a method for diagnosing and treating tumors. The method prepares cell membrane proteins from tumor tissue cells, clarifies the modification sites by mass spectrometry, modifies the molecular structure, and identifies the modified polypeptides / proteins. The method prepares the newly modified target protein or polypeptide, and uses the target protein or polypeptide to prepare antibodies for diagnosing tumors and antibodies for treating tumors. When searching the library, the modified polypeptides and proteins are found based on the amino acid sites, connected molecular sites, and modification groups of the polypeptides / proteins. The function of the modified proteins can be used to explain the biological behavior of the tumor. With a clear structure of the modified polypeptides and proteins, antibodies can be prepared. After the antibodies are prepared, they can be used for tumor diagnosis. Humanized antibodies can eliminate tumor cells due to antigen-antibody reactions, and can also carry cytotoxic drugs to kill tumors in a targeted manner.

Description

Technical Field

[0001] The present application relates to the field of biogenetic engineering technology, and specifically to a method for preparing tumor cell-specific modified polypeptides / proteins, antibodies, and tumor diagnosis and treatment methods. Background Art

[0002] Most tumors remain incurable due to the lack of tumor-specific antigens. Numerous tumor markers have been discovered, some of which are used clinically for diagnosis. However, these antigens are present not only in tumor cells but also in normal cells, making their use in diagnosis only a guideline. Chemotherapy and radiotherapy, however, cannot precisely target tumor cells due to the lack of tumor-specific antigens, leading to the destruction of both normal and tumor cells.

[0003] Based on the characteristics of tumors, after 10 years of research, a theory of tumor pathogenesis was established. The core of the theory is that tumor development is caused by the addition of new modification groups to proteins. This new modification group theory can explain tumor development, metabolic characteristics, metastasis, calcification, immortality, and immune evasion.

[0004] Based on this theory, infrared spectroscopy revealed that only tumor cells harbor this new modification group, while normal cells lack it. Further mass spectrometry confirmed the presence of this modification group and identified the modified peptides and proteins. Based on the protein's function, this could fully explain tumorigenesis, its metabolic characteristics, why it metastasizes, and why tumors evade immune responses.

[0005] By discovering the unique modifications of tumor cells, corresponding antibodies can be produced as modified peptides and proteins; with monoclonal antibodies, treatment of tumor cells will be targeted killing without affecting normal cells, making it possible to cure tumors.

[0006] After modification of specific proteins, abnormal spatial positions lead to abnormal protein functions, which in turn causes the occurrence and development of tumors. For example, tumor immune evasion functional proteins. The discovered signal-proliferation pathways and other functional protein groups are of great value for future medical applications. Corresponding antibodies can be prepared based on proteins or peptides, providing a basis for controlling cellular behavior in the future.

[0007] Therefore, it is necessary to propose a preparation method for the new modified gene protein to obtain the modified polypeptide or protein in tumor cells. Summary of the Invention

[0008] The main purpose of this application is to provide a method for preparing tumor cell-specific modified polypeptides / proteins, antibodies and tumor diagnosis and treatment methods to solve current problems.

[0009] In order to achieve the above objectives, this application provides the following technologies:

[0010] The first aspect of the present application provides a method for preparing a tumor cell-specific modified polypeptide / protein, comprising the following steps:

[0011] Cell membrane proteins were extracted from tumor tissues;

[0012] Using protein enzymatic hydrolysis method, enzymatically hydrolyze the cell membrane protein to obtain the target polypeptide;

[0013] Enriching the target polypeptide by a strong anion exchange chromatography method to obtain an enriched target polypeptide;

[0014] Perform mass spectrometry analysis and identification on the enriched target peptide to determine whether the target modification group is present;

[0015] According to the modification type and modification site, and through PD library search, the sequence of the modified gene is clarified, and a method for preparing the tumor cell-specific modified polypeptide / protein is obtained.

[0016] As an optional embodiment of the present application, optionally, extracting cell membrane proteins from tumor tissue includes:

[0017] To prepare tumor tissue, place 400-500 mg of tumor tissue in a 5 ml microcentrifuge tube, add 4 ml of cell wash solution to the tissue, vortex briefly, and discard the wash solution;

[0018] Transfer to a 2 ml tissue grinder and cut the tissue into small pieces with scissors. Add 1 ml of permeabilization buffer to the tissue and grind until a homogeneous suspension is formed.

[0019] Add 1 ml of permeabilization buffer and transfer the homogenate to a new reaction tube. Incubate at 4°C for 10 minutes with continuous mixing.

[0020] Centrifuge at 16,000 × g, 4°C for 15 minutes to pellet the permeabilized cells. Carefully remove the supernatant containing cytoplasmic proteins and transfer it to a new reaction tube.

[0021] Resuspend the pellet in 1 ml of solubilization buffer, pipette up and down to obtain a homogenous resuspension, and incubate at 4°C for 30 minutes with constant mixing.

[0022] The mixture was centrifuged at 16,000*g-4°C for 15 minutes, and the supernatant containing the soluble membrane protein and membrane-associated protein was transferred to a new protein solution sample tube to obtain the cell membrane protein.

[0023] As an optional embodiment of the present application, optionally, the extracting cell membrane protein from tumor tissue further comprises:

[0024] NanoDrop One A280 ultraviolet light was used to measure the supernatant to determine the protein content of the cell membrane protein in the supernatant.

[0025] As an optional embodiment of the present application, optionally, the protein enzymatic hydrolysis method adopts an ultrafiltration-assisted sample preparation method - FASP enzymatic hydrolysis method.

[0026] As an optional embodiment of the present application, optionally, the enzymatic hydrolysis of the cell membrane protein to obtain the target polypeptide comprises:

[0027] Add dithiothreitol (DTT) solution to the above protein solution sample test tube to obtain a solution with a final DTT concentration of 100 mM. Place the test tube in a boiling water bath for 5 minutes, then place it in a test tube rack and gradually cool it to room temperature.

[0028] Add 200 μL of UA buffer to the above solution and mix well. Transfer the mixture into a 10 kD ultrafiltration centrifuge tube and centrifuge at high speed (14000 g, 15 min). Discard the filtrate and repeat this step to obtain the supernatant.

[0029] Add 100 μL of 50 mmol / l IAA buffer, shake at 600 rpm for 1 min, react at room temperature in the dark for 30 min, centrifuge at high speed (14,000 g for 15 min), add 100 μL of UA buffer again, and centrifuge at 14,000 g for 15 min. Repeat this step twice.

[0030] Add 100 μL of 25 mM NH 4 HCO 3 solution, centrifuge (14000 g for 15 min), and repeat this step twice;

[0031] Add 40 μL of Trypsin buffer, shake at 600 rpm for 1 minute, and incubate at 37°C for 16-18 hours (1:50 mass ratio).

[0032] Replace the collection tube with a new one and centrifuge (14000g for 15 min); add 40 μL of 25 mM NH4HCO3, centrifuge at 14000g for 15 min, and collect the filtrate; wherein the filtrate contains the target protein or polypeptide.

[0033] As an optional embodiment of the present application, optionally, the enrichment of the target protein or polypeptide by a strong anion exchange chromatography method to obtain the enriched target protein or polypeptide comprises:

[0034] Prepare elution buffer A (0.05M Tris-HCl, pH = 8.5), elution buffer B (0.5M NaCl, 0.05M Tris-HCl, pH = 8.5), 20% ethanol, and 1M NaOH. Filter the elution buffer, 20% ethanol, 1M NaOH, and deionized water through a 0.45 μm filter to remove any impurities. Store the solutions at 4°C until ready to use.

[0035] Clean the AKTA protein rapid purification system, connect the AKTA, Capto HiRes Q 5 / 50 prepacked column, UV detector and collection device, and check for air tightness;

[0036] Adjust the refrigerator temperature to 4°C, add deionized water and elution buffer, balance them until the detector curve no longer changes, and adjust the flow rate to 0.2 mL / min;

[0037] The filtrate collected after the above enzymatic hydrolysis was fully dissolved in elution buffer, filtered with a 0.45 μm disposable injection filter to remove insoluble matter, and connected to the AKTA protein rapid purification system for sample loading;

[0038] After loading, add elution buffer A to wash out the unadsorbed proteins. After the flow-through peak is completely washed out, perform gradient elution with elution buffer A and elution buffer B, and collect each elution peak separately.

[0039] The peptide peaks were collected starting from 3 mAU and stored frozen at -80 degrees.

[0040] As an optional embodiment of the present application, optionally, the enriched target polypeptide is subjected to mass spectrometry analysis and identification to determine whether the target modification group is present, including:

[0041] Collect the enriched target peptides as analysis samples;

[0042] Each sample was chromatographically separated using the nano-flow HPLC liquid phase system Easy nLC;

[0043] After chromatographic separation, the samples were analyzed by mass spectrometry using a Q-Exactive mass spectrometer to identify the post-translational modification type and determine the modification site.

[0044] As an optional embodiment of the present application, optionally, according to the modification type and modification site, and after searching the PD library, the sequence of the modified gene is clarified to obtain the preparation method of the tumor cell-specific modified polypeptide / protein, comprising:

[0045] Prepare protein identification software: Mascot and Proteome Discoverer;

[0046] Protein identification software is used to search and identify the modification type and modification site and conduct quantitative analysis to clarify the type and modification site of the modification group, determine the modified molecular weight and structure of the modification group, and obtain the preparation method of the tumor cell-specific modified polypeptide / protein.

[0047] The second aspect of the present application provides an antibody, which is prepared according to the method for preparing a tumor cell-specific modified polypeptide / protein described above.

[0048] The third aspect of the present application provides a method for diagnosing and treating tumors, which utilizes the above-mentioned antibodies to prepare in vitro diagnostic kits; or utilizes the above-mentioned antibodies to eliminate tumor cells through antigen-antibody cytotoxicity.

[0049] Compared with the existing technology, this application can bring the following technical effects:

[0050] Based on the embodiment scheme of the present application, cell membrane proteins are prepared from tumor tissue cells, and after proteolysis, the target polypeptide is enriched by strong anion exchange chromatography. The polypeptide is subjected to mass spectrometry to determine the modification site, the modified molecular structure, and the correctness of the modified polypeptide / protein. When searching the library, the modified polypeptides and proteins found may be unprecedented due to the different amino acid sites, attached molecular sites, and modification groups of the polypeptide / protein. Mass spectrometry identifies the modified proteins or polypeptides and genes, and antibodies are prepared against the newly modified target proteins or polypeptides and their genes. The target proteins or polypeptides are used to prepare antibodies for tumor diagnosis and for the preparation of antibodies for tumor treatment. In addition, the function of the modified protein can be used to explain the biological behavior of the tumor, and corresponding antibodies can be prepared and used to control the biological behavior of the cell. The prepared antibodies can be used for diagnosis, and humanized antibodies can carry cytotoxic drugs to kill tumors in a targeted manner. It is also possible to eliminate tumor cells due to antigen-antibody reactions. Therefore, this method can detect modified polypeptides or proteins in tumor cells and is a method for discovering and accurately extracting modified proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The drawings that constitute part of this application are used to provide a further understanding of this application and make other features, objects and advantages of this application more apparent. The illustrative embodiment drawings of this application and their descriptions are used to explain this application and do not constitute an improper limitation of this application. In the drawings:

[0052] Figure 1 It is a schematic diagram of the implementation process of the method for preparing the tumor cell-specific modified polypeptide / protein of the present invention. DETAILED DESCRIPTION

[0053] In order to enable those skilled in the art to better understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of this application.

[0054] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequential order. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present application described here. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0055] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0056] Example 1

[0057] The first aspect of the present application provides a method for preparing a tumor cell-specific modified polypeptide / protein, comprising the following steps:

[0058] 1. Extract cell membrane proteins from tumor tissue;

[0059] 2. Using protein enzymatic hydrolysis to hydrolyze the cell membrane protein to obtain the target protein or peptide;

[0060] The FASP enzymatic hydrolysis method is preferred to hydrolyze the membrane protein into peptides; the target peptide needs to be determined based on the target modification group derived theoretically;

[0061] 3. The target polypeptide is enriched by strong anion exchange chromatography to obtain an enriched target polypeptide; enrichment of the target polypeptide allows the modification to be found in the mass spectrometry.

[0062] After enzymatic hydrolysis of membrane proteins into peptides, enrichment, reverse-phase chromatography, and mass spectrometry are performed to identify the type and site of post-translational modifications. However, these post-translationally modified proteins have low abundance and low stoichiometry. Without enrichment, they are easily masked by more abundant proteins, resulting in poor experimental results. Therefore, enrichment of the target peptide is essential to identify the modification in mass spectrometry.

[0063] In this example, strong anion exchange chromatography was used. Since the amino groups of the strong anion exchange column adsorbed onto the carboxyl groups on the polypeptide, the polypeptide was washed with a buffer solution and eluted from the exchange column under high sodium to obtain a polypeptide rich in carboxyl groups.

[0064] 4. Perform mass spectrometry analysis and identification on the enriched target protein or peptide to determine whether the target modification group is present;

[0065] Peptides can be further purified and separated by liquid chromatography before being analyzed by mass spectrometry to detect missing molecular weights and identify the presence of targeted modification groups. For proteins, mass spectrometry can not only determine the molecular weights of peptides and proteins, but also their amino acid sequences and post-translational modifications. To determine post-translational modifications, proteins are first enzymatically cleaved into peptide fragments, which are then analyzed by mass spectrometry. Mass spectrometry analysis yields information on the relative molecular masses of a series of peptide fragments.

[0066] 5. According to the modification type and modification site, and after checking the PD library, the sequence of the modification group is clarified, and the preparation method and gene of the tumor cell-specific modified polypeptide / protein are obtained.

[0067] The original data of mass spectrometry analysis were RAW files, and protein identification software Mascot2.2 and ProteomeDiscoverer1.4 were used for library identification and quantitative analysis.

[0068] The relevant parameters and descriptions are as follows: trypsin digestion, a maximum number of allowed missed cleavage sites of 2, and fixed modification of Carbamidomethyl (C);

[0069] Variable modification Oxidation (M), Acetyl (Protein N-term); primary ion mass tolerance: ±20 ppm, secondary ion mass tolerance: 0.1 Da.

[0070] Database used for querying: You can choose it by yourself;

[0071] The screening criteria for trustworthy proteins is high. The database structure is as follows:

[0072]

[0073]

[0074] For example, when searching the library, the final peptide is matched with the corresponding GO analysis of the corresponding protein and protein pathway in the library, and the variable modification Oxidation (M) is obtained, Acetyl (Protein N-term): the modified molecular weight is 72, and the amino acid is serine and threonine with the side chain hydroxyl group connected to C3H5O2.

[0075] As an optional embodiment of the present application, optionally, extracting cell membrane proteins from tumor tissue includes:

[0076] To prepare tumor tissue, place 400-500 mg of tumor tissue in a 5 ml microcentrifuge tube, add 4 ml of cell wash solution to the tissue, vortex briefly, and discard the wash solution;

[0077] Transfer to a 2 ml tissue grinder and cut the tissue into small pieces with scissors. Add 1 ml of permeabilization buffer to the tissue and grind until a homogeneous suspension is formed.

[0078] Add 1 ml of permeabilization buffer and transfer the homogenate to a new reaction tube. Incubate at 4°C for 10 minutes with continuous mixing.

[0079] Centrifuge at 16,000 × g, 4°C for 15 minutes to pellet the permeabilized cells. Carefully remove the supernatant containing cytoplasmic proteins and transfer it to a new reaction tube.

[0080] Resuspend the pellet in 1 ml of solubilization buffer, pipette up and down to obtain a homogenous resuspension, and incubate at 4°C for 30 minutes with constant mixing.

[0081] The mixture was centrifuged at 16,000*g-4°C for 15 minutes, and the supernatant containing the soluble membrane protein and membrane-associated protein was transferred to a new protein solution sample tube to obtain the cell membrane protein.

[0082] As an optional embodiment of the present application, optionally, the extracting cell membrane protein from tumor tissue further comprises:

[0083] NanoDrop One A280 ultraviolet light was used to measure the supernatant to determine the protein content of the cell membrane protein in the supernatant.

[0084] As an optional embodiment of the present application, optionally, the protein enzymatic hydrolysis method adopts an ultrafiltration-assisted sample preparation method - FASP enzymatic hydrolysis method.

[0085] As an optional embodiment of the present application, optionally, the enzymatic hydrolysis of the cell membrane protein to obtain the target polypeptide comprises:

[0086] Add dithiothreitol (DTT) solution to the above protein solution sample test tube to obtain a solution with a final DTT concentration of 100 mM. Place the test tube in a boiling water bath for 5 minutes, then place it in a test tube rack and gradually cool it to room temperature.

[0087] Add 200 μL of UA buffer to the above solution and mix well. Transfer the mixture into a 10 kD ultrafiltration centrifuge tube and centrifuge at high speed (14000 g, 15 min). Discard the filtrate and repeat this step to obtain the supernatant.

[0088] Add 100 μL of 50 mmol / l IAA buffer, shake at 600 rpm for 1 min, react at room temperature in the dark for 30 min, centrifuge at high speed (14,000 g for 15 min), add 100 μL of UA buffer again, and centrifuge at 14,000 g for 15 min. Repeat this step twice.

[0089] Add 100 μL of 25 mM NH 4 HCO 3 solution, centrifuge (14000 g for 15 min), and repeat this step twice;

[0090] Add 40 μL of Trypsin buffer, shake at 600 rpm for 1 minute, and incubate at 37°C for 16-18 hours (1:50 mass ratio).

[0091] Replace the collection tube with a new one and centrifuge (14000g for 15 min); add 40 μL of 25 mM NH4HCO3, centrifuge at 14000g for 15 min, and collect the filtrate; wherein the filtrate contains the target protein or polypeptide.

[0092] As an optional embodiment of the present application, optionally, the enrichment treatment of the target protein or polypeptide to obtain the enriched target polypeptide includes:

[0093] Prepare elution buffer A (0.05M Tris-HCl, pH = 8.5), elution buffer B (0.5M NaCl, 0.05M Tris-HCl, pH = 8.5), 20% ethanol, and 1M NaOH. Filter the elution buffer, 20% ethanol, 1M NaOH, and deionized water through a 0.45 μm filter to remove any impurities. Store the solutions at 4°C until ready to use.

[0094] Clean the AKTA protein rapid purification system, connect the AKTA, Capto HiRes Q 5 / 50 prepacked column, UV detector and collection device, and check for air tightness;

[0095] Adjust the refrigerator temperature to 4°C, add deionized water and elution buffer, balance them until the detector curve no longer changes, and adjust the flow rate to 0.2 mL / min;

[0096] The filtrate collected after the above enzymatic hydrolysis was fully dissolved in elution buffer, filtered with a 0.45 μm disposable injection filter to remove insoluble matter, and connected to the AKTA protein rapid purification system for sample loading;

[0097] After loading, add elution buffer A to wash out unadsorbed proteins. Once the flow-through peak has completely washed out, perform a gradient elution using elution buffer A and then elution buffer B. Collect each elution peak, starting at 3 mAU. Store frozen at -80°C. After lyophilization, reconstitute the peptides in 40 μL of 0.1% formic acid solution and quantify the peptides using the OD280 protein assay.

[0098] Enrichment:

[0099] Proteins are dichroic, with varying charges at different pH values. Currently, proteins are known to be weakly acidic, with isoelectric points ranging from 3 to 6. While simple protein purification yields weakly acidic proteins, it cannot be completely certain that no amino acids in basic proteins have been modified. Therefore, enzymatic analysis after protein purification can result in the loss of modified peptides. In a solution at approximately pH 8.5, the N-terminal amino group and any histidine residues in the peptide produced after enzymatic hydrolysis are not protonated but rather exist in a neutral form. However, Asp, Glu, and tyrosine residues in the peptide are all anionic, with lysine partially protonated and arginine fully protonated. At this pH, peptides rich in Asp, Glu, and tyrosine residues are readily retained by ion exchange. Therefore, strong anion exchange chromatography is recommended after membrane proteolysis to enrich for -COOH-containing peptides. The target peptides have modified amino acid side chains, resulting in terminal -COOH groups, which are anionic at pH 8.5. Therefore, strong anion exchange chromatography is recommended.

[0100] As an optional embodiment of the present application, optionally, the enriched target polypeptide is subjected to mass spectrometry analysis and identification to determine whether the target modification group is present, including:

[0101] Collect the enriched target peptides as analysis samples;

[0102] Each sample was chromatographically separated using the nano-flow HPLC liquid phase system Easy nLC;

[0103] After chromatographic separation, the samples were analyzed by mass spectrometry using a Q-Exactive mass spectrometer to identify the post-translational modification type and determine the modification site.

[0104] For proteins, mass spectrometry can not only determine the molecular weight of peptides and proteins, but also the amino acid sequence and post-translational modification of peptides. For post-translational modification of proteins, the protein is first enzymatically cleaved into peptides and then analyzed by mass spectrometry; through mass spectrometry analysis, the relative molecular mass information of a series of peptides is obtained. For a specific peptide, its sequence information and molecular weight are certain in the absence of any post-translational modification. When it undergoes a certain post-translational modification, it is found during the mass spectrometry detection process that the molecular weight of some peptides just increases or decreases the molecular weight of the modification group. In this case, it can be assumed that this peptide has undergone a new modification. Secondary confirmation through secondary or multi-stage mass spectrometry can achieve post-translational modification type identification and modification site analysis.

[0105] As an optional embodiment of the present application, optionally, according to the modification type and modification site, and after searching the PD library, the sequence of the modification group is clarified to obtain the preparation method of the tumor cell-specific modified polypeptide / protein, comprising:

[0106] Prepare protein identification software: Mascot and Proteome Discoverer;

[0107] Protein identification software is used to search and identify the modification type and modification site and conduct quantitative analysis to clarify the type and modification site of the modification group, determine the modified molecular weight and structure of the modification group, and obtain the preparation method of the tumor cell-specific modified polypeptide / protein.

[0108] Example 2

[0109] The second aspect of the present application provides an antibody, which is prepared according to the method for preparing a tumor cell-specific modified polypeptide / protein described above.

[0110] Once the modified peptide is obtained, the modified protein can be searched and catalogued for antibodies. These antibodies can then be used to prepare in vitro diagnostic kits. Humanized antibodies can carry cytotoxic drugs for targeted tumor killing, or they can eliminate tumor cells through antigen-antibody reactions. There are no restrictions on the method for preparing antibodies. For example, a method for preparing tumor cell-specific modified peptides / proteins (antigens) can be used to generate humanized antibodies after inoculation.

[0111] Because this modified polypeptide / protein is not found in normal cells but only in tumor cells, it is like penicillin treating bacteria. Only bacteria have cell walls, but the human body does not. Penicillin only targets bacteria and has little effect on the human body.

[0112] Therefore, antibodies produced by targeting this new amino acid modification will be completely new, making tumor-specific antibodies a great option for curing tumors. Antibodies produced by targeting modified protein groups can control cell behavior and provide the potential for biomimetic tissue and organ regeneration.

[0113] Example 3

[0114] The third aspect of the present application provides a method for diagnosing and treating tumors, in which the antibodies prepared by the above-mentioned method are used in in vitro diagnostic kits: including enzyme-linked immunosorbent assay, chemiluminescence assay, colloidal gold assay, etc. to prepare reagents; or the antibodies described above are used to eliminate tumor cells through antigen-antibody cytotoxicity.

[0115] Example 4

[0116] Based on Example 1, in addition to isolating and extracting modified proteins from the cell membrane, new modifications to proteins / peptides should theoretically occur at O-junctions within the Golgi apparatus. However, since lactic acid products cannot enter the Golgi apparatus, they should be located on the endoplasmic reticulum. Modified proteins / peptides are also present in the cytoplasm, albeit in smaller quantities. Antibodies can also be generated against these modified peptides or proteins.

[0117] Therefore, this embodiment also provides a separation method for extracting modified proteins in the cytoplasm.

[0118] To prepare tumor tissue, place 400-500 mg of tumor tissue in a 5 ml microcentrifuge tube, add 4 ml of cell wash solution to the tissue, vortex briefly, and discard the wash solution;

[0119] Transfer to a 2 ml tissue grinder and cut the tissue into small pieces with scissors. Add 1 ml of permeabilization buffer to the tissue and grind until a homogeneous suspension is formed.

[0120] Add 1 ml of permeabilization buffer and transfer the homogenate to a new reaction tube. Incubate at 4°C for 10 minutes with continuous mixing.

[0121] Permeabilized cells were pelleted by centrifugation at 16,000 × g at 4°C for 15 minutes. The supernatant containing cytosolic proteins was transferred to a new reaction tube. Protein content was determined using a NanoDrop One A280 UV spectrophotometer.

[0122] Using protein enzymatic hydrolysis method, enzymatically hydrolyze the cell cytoplasm protein to obtain the target polypeptide;

[0123] Enriching the target polypeptide by a strong anion exchange chromatography method to obtain an enriched target polypeptide;

[0124] Perform mass spectrometry analysis and identification on the enriched target peptide to determine whether the target modification group is present;

[0125] According to the modification type and modification site, and through PD library search, the sequence of the modified gene is clarified, and a method for preparing the tumor cell-specific modified polypeptide / protein is obtained.

[0126] The above method does not require:

[0127] Resuspend the pellet in 1 ml of solubilization buffer, pipette up and down to obtain a homogenous resuspension, and incubate at 4°C for 30 minutes with constant mixing.

[0128] The mixture was centrifuged at 16,000*g-4°C for 15 minutes, and the supernatant containing the soluble membrane protein and membrane-associated protein was transferred to a new protein solution sample tube to obtain the cell membrane protein.

[0129] The steps of Example 1 can be combined for comparison.

[0130] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for preparing a tumor cell-specific modified polypeptide or protein, characterized in that: The steps include: Cell membrane proteins were extracted from tumor tissues; The cell membrane protein is enzymatically hydrolyzed to obtain the target polypeptide, comprising: Add dithiothreitol (DTT) solution to the above protein solution sample test tube to obtain a solution with a final DTT concentration of 100 mM. Place the test tube in a boiling water bath for 5 minutes, then place it in a test tube rack and gradually cool it to room temperature. Add 200 μL of UA buffer to the above solution and mix well. Transfer the mixture into a 10kD ultrafiltration centrifuge tube and centrifuge at high speed. Discard the filtrate and repeat this step to obtain the supernatant. Add 100 μL of 50 mmol / l IAA buffer, shake at 600 rpm for 1 min, react at room temperature in the dark for 30 min, centrifuge at high speed, add 100 μL of UA buffer again and centrifuge, repeat this step twice; Add 100 μL of 25 mM NH 4 HCO 3 solution, centrifuge, and repeat this step twice; Add 40 μL of Trypsin buffer, shake at 600 rpm for 1 min, and incubate at 37°C for 16-18 h. Replace the collection tube with a new one and centrifuge; add 40 μL of 25 mM NH4HCO3, centrifuge at 14000*g for 15 min, and collect the filtrate; wherein the filtrate contains the target polypeptide; The target polypeptide was enriched by a strong anion exchange chromatography method to obtain the enriched target polypeptide, including: preparing elution buffer A, elution buffer B, 20% ethanol and 1M NaOH, wherein: elution buffer A is 0.05M Tris-HCl, pH=8.5; elution buffer B is 0.5 M NaCl, 0.05 M Tris-HCl, pH=8.5; elution buffer, 20% ethanol, 1M NaOH and deionized water were filtered using a 0.45 μm filter membrane to remove a small amount of impurities; the filtered and impurity-removed solution was placed at 4° C. for use; the AKTA protein rapid purification system was cleaned, and the AKTA and Capto HiRes Q 5 / 50 were connected. Pre-install the column, UV detector and collection device, and check the air tightness; adjust the refrigerator temperature to 4°C, connect deionized water and elution buffer in turn, balance until the detector curve no longer changes, and adjust the flow rate to 0.2mL / min; fully dissolve the filtrate collected after the above enzymatic hydrolysis with elution buffer, filter with a 0.45μm disposable injection filter to remove insoluble matter, and connect to the AKTA protein rapid purification system for loading; after the loading is completed, connect elution buffer A to wash out the unadsorbed protein, and after the flow-through peak is completely washed out, perform gradient elution with elution buffer A and elution buffer B, and collect each elution peak separately; collect the polypeptide peak starting from 3mAU and freeze it at -80 degrees; the polypeptide produced after enzymatic hydrolysis is in a solution with a pH of about 8.5, and the N-terminal amino group on the polypeptide and any histidine in the peptide are not protonated, but exist in a neutral form, while the Asp, Glu and tyrosine residues in the peptide are all anions, Lysine is partially protonated, while arginine is fully protonated. At this pH, peptides rich in Asp, Glu, and tyrosine residues are easily retained by ion exchange. Therefore, strong anion exchange chromatography after membrane proteolysis is selected to enrich peptides containing -COOH. The amino acid side chains of the target peptide are modified, and the terminal -COOH group is anionic in a pH 8.5 solution. Perform mass spectrometry analysis and identification on the enriched target peptide to determine whether the target modification group is present; Based on the modification type and modification site, and after PD library search, the sequence of the modified gene was clarified, and the tumor cell-specific modified polypeptide or protein was obtained: variable modification Oxidation (M), Acetyl (Protein N-term): modified molecular weight 72, amino acids serine and threonine side chain hydroxyl connected to C3H5O2; primary ion mass tolerance: ± 20 ppm; secondary ion mass tolerance: 0.1Da.

2. The method for preparing a tumor cell-specific modified polypeptide or protein according to claim 1, wherein: The cell membrane protein is extracted from the tumor tissue, comprising: To prepare tumor tissue, place 400-500 mg of tumor tissue in a 5 ml microcentrifuge tube, add 4 ml of cell wash solution to the tissue, vortex briefly, and discard the wash solution; Transfer to a 2 ml tissue grinder and cut the tissue into small pieces with scissors. Add 1 ml of permeabilization buffer to the tissue and grind until a homogeneous suspension is formed. Add 1 ml of permeabilization buffer and transfer the homogenate to a new reaction tube. Incubate at 4°C for 10 minutes with continuous mixing. Centrifuge at 16,000 × g, 4°C for 15 minutes to pellet the permeabilized cells. Carefully remove the supernatant containing cytoplasmic proteins and transfer it to a new reaction tube. Resuspend the pellet in 1 ml of solubilization buffer, pipette up and down to obtain a homogenous resuspension, and incubate at 4°C for 30 minutes with constant mixing. The mixture was centrifuged at 16,000*g and 4 degrees for 15 minutes, and the supernatant containing soluble membrane proteins and membrane-associated proteins was transferred to a new protein solution sample tube to obtain the cell membrane protein.

3. The method for preparing a tumor cell-specific modified polypeptide or protein according to claim 2, wherein: The method of extracting cell membrane proteins from tumor tissues further comprises: The supernatant was measured using NanoDrop One A280 ultraviolet light to determine the protein content of the cell membrane protein in the supernatant.

4. The method for preparing a tumor cell-specific modified polypeptide or protein according to claim 1, wherein: The protein enzymatic hydrolysis method adopts an ultrafiltration-assisted sample preparation method---FASP enzymatic hydrolysis method.

5. The method for preparing a tumor cell-specific modified polypeptide or protein according to claim 1, wherein: Perform mass spectrometry analysis and identification on the enriched target peptide to determine whether there are target modification groups, including: Collect the enriched target peptides as analysis samples; Each sample was chromatographically separated using the nano-flow HPLC liquid phase system Easy nLC; After chromatographic separation, the samples were analyzed by mass spectrometry using a Q-Exactive mass spectrometer to identify the post-translational modification type and determine the modification site.

6. The method for preparing a tumor cell-specific modified polypeptide or protein according to claim 1, wherein: According to the modification type and modification site, and after searching the PD database, the sequence of the modified gene is determined, and the preparation method of the tumor cell-specific modified polypeptide or protein is obtained, including: Prepare protein identification software: Mascot and Proteome Discoverer; Protein identification software is used to search and identify the modification type and modification site and conduct quantitative analysis to clarify the type and modification site of the modification group, determine the modified molecular weight and structure of the modification group, and obtain the preparation method of the tumor cell-specific modified polypeptide or protein.

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