Probe for capturing response protein of drug damage DNA (Deoxyribonucleic Acid) as well as preparation method and application of probe

Abstract

The invention relates to the field of protein enrichment, separation and detection, and discloses a probe for capturing response protein of drug damage DNA, the probe comprises the following elements connected in series: (nuclear localization signal-BRD2) n-biotin ligase, and n is an integer from 1 to 4. The probe provided by the invention is good in affinity, the quantity of the response protein for capturing the drug damage DNA can be increased, and the specificity of the response protein for capturing the drug damage DNA can also be considered. The method for capturing the response protein of the drug-damaged DNA is simple, convenient and rapid, can be used for capturing and identifying the response protein in any drug cells which cause DNA damage through covalent bond or coordination bond combination, and also provides a new idea for capturing the response protein of the drug-damaged DNA.

Description

Technical Field

[0001] The present invention relates to the field of protein enrichment, separation and detection, and in particular to a probe for capturing a protein responsive to drug-damaged DNA, and a preparation method and application thereof. Background Art

[0002] Since the anti-tumor activity of cisplatin was discovered by Professor Rosenberg and others at Michigan State University in 1969, cisplatin has become one of the most widely used anti-cancer drugs in clinical practice. The platinum anti-cancer drugs it represents are the model of cytotoxic cancer chemotherapy drugs. Second-generation platinum drugs such as carboplatin and oxaliplatin have also been put into clinical use. A large number of studies have shown that the ultimate target of cisplatin and its analogs is DNA in the cell nucleus. After cisplatin enters the human body, it enters the cell by passive diffusion and undergoes hydrolysis. The generated hydrate is transported to the cell nucleus and then coordinates with guanine and adenine on DNA to form a platinum-DNA complex, of which 1,2-intrachain cross-linked complexes account for about 90%. By forming cross-linked complexes with DNA, cisplatin can hinder DNA replication and transcription, induce cell apoptosis, and thus produce anti-tumor activity. When the DNA in the cell nucleus is damaged by cisplatin, the cell will inevitably respond to it. Platinum-DNA response proteins in cells mainly include proteins involved in the damage repair process, such as hMutS, DNA-dependent protein kinase, and high-mobility group proteins, such as HMGB1. HMGB1 is a DNA-binding protein that can specifically interact with cisplatin-crosslinked DNA. Other proteins, such as TATA-binding protein (TBP), poly (ADP-ribose) polymerase 1 (PARP-1) and p53 protein, have also been found to recognize cisplatin-damaged DNA. These platinum-DNA response proteins communicate with each other in structure and function, which has an important impact on the anti-tumor activity of cisplatin. Therefore, studying the response of cells to DNA damage by platinum drugs at the molecular level is conducive to a deeper understanding of the mechanism of action of cytotoxic anticancer drugs, improving the efficacy of existing drugs and optimizing the design of new metal anticancer drugs.

[0003] At present, the methods for capturing platinum-damaged DNA response proteins include affinity enrichment technology, photocrosslinking technology, and nano-affinity probe capture technology. For example, cisplatin-ctDNA modified microspheres are used to enrich cell lysate by affinity, but this method is complicated to operate and requires a large amount of DNA; there are also methods to couple photoreactive groups to cisplatin to form a stable DNA-Pt-protein complex through photocrosslinking, but the structural changes to cisplatin will affect the binding of cisplatin to DNA and the binding of protein to platinum-DNA adducts. At the same time, directly coupling cisplatin to protein may cause more false positives. There is still a need to develop new methods for capturing platinum-damaged DNA response proteins. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems existing in the prior art and provide a probe for capturing a protein responsive to drug-damaged DNA. The probe can specifically capture a protein responsive to damaged DNA and capture a large number of proteins.

[0005] After drugs such as cisplatin cause DNA double-strand break damage (DSB), histone acetylation will also recruit related repair proteins to the vicinity of the damage site. For example, H4K8 acetylation will recruit proteins such as 53BP1, RAD51, KU70 and KU80 to the vicinity of the damage site. Based on this, the inventors of the present invention have found in their research that by using a proximity labeling technique mediated by a specific composition of probes, the neighboring proteins of the histone acetylation site can be analyzed to obtain information on the genotoxic drug response proteins in the cell, and the number of captured proteins can be increased while taking into account the specificity of the captured proteins. Thus, the present invention is obtained.

[0006] In order to achieve the above object, the present invention provides a probe for capturing a protein responsive to drug-damaged DNA in a first aspect, wherein the probe comprises the following elements (nuclear localization signal sequence-BRD2) connected in series: n -Biotin ligase, wherein n is an integer of 1-4.

[0007] The second aspect of the present invention provides a method for preparing the probe, wherein the method comprises introducing a plasmid expressing the probe into a target cell.

[0008] The third aspect of the present invention provides the use of the probe or the probe prepared by the method in capturing and identifying proteins responsive to drug-damaged DNA.

[0009] A fourth aspect of the present invention provides a method for capturing a protein responsive to drug-damaged DNA, wherein the method comprises the following steps:

[0010] (1) introducing a plasmid expressing the probe described in the first aspect of the present invention into a target cell, and then contacting the target cell with biotin and a drug so that the biotin binds to the probe;

[0011] (2) extracting nuclear proteins from target cells, separating captured biotinylated proteins, and then performing mass spectrometry detection;

[0012] (3) Quantitative analysis of the identified proteins was performed to exclude nonspecifically bound proteins and intracellular self-biotinylated proteins, and to screen out proteins that respond to drug-damaged DNA.

[0013] The probe provided by the present invention has good affinity, can increase the number of captured drug-damaged DNA response proteins, and can also take into account the specificity of captured drug-damaged DNA response proteins. The method for capturing drug-damaged DNA response proteins provided by the present invention is simple and rapid, and can be used for capturing and identifying any drug intracellular response proteins that cause DNA damage through covalent or coordination bond binding, and also provides a new idea for capturing drug-damaged DNA response proteins.

[0014] Furthermore, the method for capturing the response protein to drug-damaged DNA provided by the present invention is simple, rapid, mild, and saves time and effort. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A process for capturing and identifying proteins that respond to drug-damaged DNA.

[0016] Figure 2 These are fluorescent images of living cells after negative (left) and positive (right) probes were transfected into cells according to the method of Example 2.

[0017] Figure 3 The sample solution after enrichment of biotinylated proteins with streptavidin magnetic beads according to the method of Example 3, Coomassie brilliant blue staining of the flow-through solution and streptavidin magnetic beads-enriched proteins (left) and immunoblotting results using streptavidin coupled to horseradish peroxidase (right).

[0018] Figure 4 The negative and positive probes were transfected according to the method of Example 4 and the mass spectrometry results of the enriched proteins were detected.

[0019] Figure 5 The mass spectrometry results of the control group proteins transfected with biotin-labeled probes and added with cisplatin and without cisplatin according to the method of Example 5 are shown in the figure. represent up-regulated and down-regulated proteins, respectively.

[0020] Figure 6 Gene Ontology (GO) analysis of all identified biotinylated proteins shows the results of enrichment in cellular location (Cellular Component) and molecular function (Molecular Function).

[0021] Figure 7 The GO analysis of negative response proteins was enriched in biological processes.

[0022] Figure 8When using western blotting (WB) to verify the key proteins identified in Example 5, the WB results of the control group (no cisplatin added) and the cisplatin group nuclear protein and the XRCC5 (KU80) and NPM1 proteins captured in the protein using streptavidin magnetic beads. From left to right are the control group and the cisplatin group; from top to bottom are the XRCC5 (KU80) and NPM1 proteins captured by streptavidin magnetic beads, the XRCC5 (KU80) and NPM1 proteins in the nuclear protein, and the internal reference lamin A protein. DETAILED DESCRIPTION

[0023] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0024] In the present invention, unless otherwise specified, the term "drug-damaged DNA" refers to an adduct formed when at least one strand of the double-stranded DNA is covalently or coordinately bound to the chemical substance after the double-stranded DNA contacts the chemical substance that can covalently or coordinately bind to the DNA, that is, the double-stranded DNA covalently or coordinately bound to the chemical substance, and this binding will cause a change in the conformation of the double-stranded DNA but will not change the base sequence of the DNA; "drug-damaged DNA response protein" refers to a protein that can specifically bind to drug-damaged DNA.

[0025] The first aspect of the present invention provides a probe for capturing a protein responsive to drug-damaged DNA, wherein the probe comprises the following elements (nuclear localization signal sequence-BRD2) connected in series: n -biotin ligase, wherein n is an integer of 1-4 (e.g., any one of 1, 2, 3 or 4). The inventors of the present invention have found through in-depth research that the probe having the above structure has high affinity, can capture a larger number of drug-damaged DNA response proteins, and has good specificity. Moreover, the inventors have found that, unlike conventional methods, when 1-4 BRD2s separated by signal peptides are used as the recognition elements of the probe in the present invention, the specificity of the captured drug-damaged DNA response proteins is better and the number is larger. When n is an integer of 2-4, the number and specificity of the captured response proteins can be further improved.

[0026] The amino acid sequence of BRD2 (Bromodomain-containing protein 2) is shown in SEQ ID NO: 2.

[0027] In the present invention, in order to facilitate the tracking and detection of the protein or probe bound by the probe, the probe may further include a marker protein, preferably a fluorescent protein, and the fluorescent protein may be a fluorescent protein conventionally used in the art, for example, may be selected from at least one of green fluorescent protein, enhanced green fluorescent protein, yellow fluorescent protein, and red fluorescent protein. In some specific embodiments of the present invention, green fluorescent protein EGFP is used as an example for description, but it should not be regarded as limiting the present invention.

[0028] In the present invention, preferably, the marker protein is linked to the biotin ligase.

[0029] In the present invention, in order to increase the number and specificity of the captured drug-damaged DNA response proteins, preferably, the nuclear localization signal sequence is selected from at least one of a single nuclear localization signal (MP-NLS), a bipartite nuclear localization signal (BP-NLS) and a proline tyrosine nuclear localization signal (PY-NLS), and more preferably at least one of MP-NLS. More preferably, the nuclear localization signal sequence is the T antigen sequence of the SV40 virus in MP-NLS, and the amino acid sequence is shown in SEQ ID NO: 1.

[0030] In the present invention, preferably, the loose sequence A consists of 10-13 (for example, 10, 11, 12 or 13) polypeptide fragments GGGGS. More preferably, the amino acid sequence of the loose sequence A is as shown in SEQ ID NO:3.

[0031] In the present invention, preferably, the biotin ligase is connected to the marker protein via a loose sequence B; more preferably, the loose sequence B consists of 1-3 (for example, 1, 2 or 3) polypeptide fragments EAAAK, and further preferably, the amino acid sequence of the loose sequence B is as shown in SEQ ID NO: 4.

[0032] In the present invention, the biotin ligase is a biotin ligase conventionally used in the art, for example, it can be at least one selected from the group consisting of the biotin ligase BirA in Escherichia coli, the R118G mutant BioID of BirA, the R40G mutant BioID2 of the thermophilic bacteria biotin ligase, the R118S mutant TurboID of BirA and the Bacillus subtilis biotin ligase BASU. In some preferred embodiments of the present invention, the biotin ligase is BASU.

[0033] The second aspect of the present invention provides a method for preparing the probe, wherein the method comprises introducing a plasmid expressing the probe into a target cell. Any starting plasmid in the art that can be used to construct a recombinant plasmid for expressing an exogenous gene in a target cell such as an engineered bacterium to obtain a target protein can be used to construct the plasmid expressing the probe of the present invention.

[0034] In some preferred embodiments of the present invention, the nucleotide sequence of the plasmid is shown in SEQ ID NO: 5. Among them, the coding sequence of the nuclear localization signal sequence is shown in nucleotides 1 to 21; the coding sequence of the recognition element BRD2 for recognizing histone modification is shown in nucleotides 43 to 609; the coding sequence of the loose sequence A is shown in nucleotides 1198 to 1404; the coding sequence of the biotin ligase BASU is shown in nucleotides 1405 to 2184; the coding sequence of the loose sequence B is shown in nucleotides 2185 to 2220; and the coding sequence of the green fluorescent protein is shown in nucleotides 2251 to 2970.

[0035] In the present invention, the target cell may be a commonly used target cell in the art, and the target cell may be a prokaryotic cell or a eukaryotic cell, as long as the probe can be expressed. In the present invention, human non-small cell lung cancer cells are used as an example for illustration, but this should not be regarded as limiting the present invention.

[0036] The method of introduction is not particularly limited, for example, it can be a chemical transformation method (such as calcium phosphate coprecipitation method, vector transfection method, etc.), electroporation method, microinjection method, gene gun method, protoplast transfection or virus-mediated transfection method. In some preferred embodiments of the present invention, the amount of the plasmid is 10-50 μg per 100 μL of the target cells, and more preferably 20-40 μg.

[0037] In the present invention, in order to make the target cells express more probes, the target cells can be cultured after introduction, and the culture time can be selected within a wide range, such as 12-48 hours. In some preferred embodiments, the culture time is 20-36 hours.

[0038] In the present invention, in order to obtain more of the plasmid, the plasmid can also be amplified, and the amplification method comprises the following steps:

[0039] (a) transforming the plasmid into competent cells and culturing them on a medium plate containing antibiotics;

[0040] (b) picking up the monoclonal colonies grown in step (a) and further amplifying them in a liquid culture medium containing the same antibiotics as in step (a);

[0041] (c) Bacteria were separated from the culture medium in (b) and the plasmid was extracted.

[0042] In the present invention, the competent cells in step (a) can be any cells in the most suitable physiological state for taking up and accommodating foreign DNA, such as JM109, DH50, BL21, TOP10, Stbl3, DH10B and DH5α, preferably DH5α cells. The volume of the liquid culture medium inoculated with the monoclonal colony in step (b) can be 15-1000mL, preferably 800-950mL; the culture time is 12-24h, preferably 12-18h. The antibiotics in step (a) depend on the antibiotics on the plasmid vector where the probe is located, such as ampicillin and kanamycin.

[0043] The third aspect of the present invention provides the use of the probe or the probe prepared by the method in capturing and identifying proteins responsive to drug-damaged DNA.

[0044] In the present invention, the drug can be any drug that causes DNA damage (e.g., changes in DNA conformation) through covalent bonding or coordination bonding, for example, a platinum anti-tumor compound among metal anti-tumor drugs, with the general formula [Pt(Ⅱ)A m B n ]; wherein A is a monodentate amino (amine) ligand or a bidentate amine ligand, such as NH3 or NH2C2H4NH2; B is a reaction leaving group, which is a monodentate anionic ligand or a bidentate anionic ligand, wherein the monodentate anionic ligand is preferably Cl-, and the bidentate anionic ligand is preferably an oxalic acid dianion; the values ​​of m and n ultimately satisfy the coordination number of the central atom Pt (II) as 4, for example, if A and B are both monodentate ligands, then m+n=4, if A and B are both bidentate ligands, then m+n=2. It should be noted that in drug-damaged double-stranded DNA, the binding modes of various conventional chemical substances to double-stranded DNA are well known to those skilled in the art and will not be described in detail here.

[0045] A fourth aspect of the present invention provides a method for capturing a protein responsive to drug-damaged DNA, wherein the method comprises the following steps:

[0046] (1) introducing a plasmid expressing the probe described in the first aspect of the present invention into a target cell, and then contacting the target cell with biotin and a drug so that the biotin binds to the probe;

[0047] (2) extracting nuclear proteins from target cells, separating captured biotinylated proteins, and performing mass spectrometry on these proteins;

[0048] (3) Quantitative analysis of the identified proteins was performed to exclude nonspecifically bound proteins and intracellular self-biotinylated proteins, and to screen out proteins that respond to drug-damaged DNA.

[0049] In the present invention, the target cells may be any cells that have DNA damage after drug treatment, including but not limited to tumor cells (eg, human non-small cell lung cancer cells).

[0050] In the present invention, in step (1), the amount of the plasmid can be selected within a wide range, as long as the target cells can express the probe. In order to increase the number of captured response proteins, preferably, relative to 1×10 6 The amount of the plasmid used is 20-50 μg, preferably 35-45 μg.

[0051] Preferably, relative to 40 μg of plasmid, the amount of biotin used is 0.5-100 μM (for example, it can be 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM and any range composed of any of the above values ​​and any value within the range), more preferably 40-70 μM.

[0052] In the present invention, the contact conditions include a growth temperature suitable for the target cells, and the contact time can be selected within a wide range as long as the biotin can bind to the probe. Preferably, under the conditions of 40 μg plasmid and 0.5-100 μM biotin, the contact time is 12-36 h, more preferably 16-24 h.

[0053] According to the present invention, after the biotin is bound to the probe, the nuclear protein of the target cell can be extracted, and then the biotinylated protein in the nuclear protein can be separated and enriched by using magnetic beads that specifically bind to the biotinylated protein.

[0054] In the present invention, the biotinylated protein-specific binding magnetic beads may be magnetic beads with any label that can specifically bind to biotin-modified proteins, such as magnetic beads with streptavidin labels, magnetic beads with avidin labels, or magnetic beads with biotin antibodies.

[0055] In the present invention, the separation and enrichment method can be a method for separating and enriching proteins commonly used in the art, for example, the protein can be first contacted with magnetic beads that specifically bind to biotinylated proteins, and then the captured biotinylated proteins are separated by external magnetic field attraction, and finally the protein is washed and collected. Wherein, the contact conditions can be 0.5-1.5 mg of magnetic beads for every 2 mg of biotinylated protein, preferably 1 mg; contact at 25°C for 1-4h, or contact at 4°C for 12-24h, preferably 12-24h, and more preferably contact at 4°C for 16-18h. The external magnetic field can attract the streptavidin magnetic beads with captured biotinylated proteins to the bottom of the reaction vessel, and the supernatant can be discarded to obtain the captured biotinylated proteins. The attraction time can be adjusted according to the actual situation, preferably, the attraction time is 0.1-3min. Wherein, the external magnetic field can be formed by a magnet. The washing liquid for washing can be a 0.01-0.5 mol / L glycine solution or a 0.1-0.5 mol / L formic acid solution, preferably a 0.01-0.5 mol / L glycine solution, and more preferably a 0.05-0.2 mol / L glycine solution.

[0056] According to the present invention, in step (3), in order to accurately exclude non-specifically bound proteins and intracellular self-biotinylated proteins, a probe not containing BRD2 can be used as a control group, and the captured biotinylated proteins can be separated according to the methods of steps (1) and (2). These proteins are non-specifically bound proteins and intracellular self-biotinylated proteins. Non-specifically bound proteins refer to proteins bound to magnetic beads through adsorption and the like.

[0057] According to the present invention, the method for identifying the captured protein can be by eluting the captured protein from the probe, and then analyzing and identifying the eluted protein by means known in the art, and finally screening out the response protein of drug-damaged DNA. Preferably, the captured protein can be eluted from the probe using a 0.01-0.5mol / L glycine solution or a 0.1-0.5mol / L formic acid solution, and then the captured protein is enzymatically hydrolyzed into peptides using trypsin, and then nanoliter liquid-mass spectrometry test is performed, and then proteomics data analysis software is used to identify and relatively quantitatively analyze the protein. The specific operation steps are well known to those skilled in the art and will not be repeated here.

[0058] According to the present invention, in order to accurately identify proteins that respond to drug-damaged DNA, cells that have not been treated with drugs can be used as controls, and proteins that only bind to undamaged DNA are captured according to the above method to exclude proteins that bind to undamaged DNA, thereby obtaining captured proteins that respond to drug-damaged DNA; the captured proteins that respond to drug-damaged DNA are identified by mass spectrometry-based quantitative proteomics, and the relative quantitative ratio of proteins that respond to drug-damaged DNA to proteins that bind to undamaged DNA is obtained, wherein proteins with significantly different abundances are proteins that specifically respond to drug-damaged DNA, and the identified proteins that specifically respond to drug-damaged DNA are verified by biochemical and molecular biological methods.

[0059] In the present invention, preferably, proteins with a relative quantitative p value < 0.05 and a relative quantitative ratio ≥ 1.20 or ≤ 0.83 are considered to be specific response proteins to drug-damaged DNA.

[0060] The present invention will be described in detail below through examples.

[0061] In the following embodiments:

[0062] Cisplatin was purchased from Beijing Coupling Technology Co., Ltd.

[0063] DH5α cells were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0064] A549 cells were purchased from the National Laboratory Cell Resource Sharing Service Platform (resource number 1101HUM-PUMC000002). The composition of the culture medium used for cell culture was: DMEM (high glucose) supplemented with 1 wt % antibiotics (100 U / mL penicillin and 100 mg / mL streptomycin) and 10 vol % fetal bovine serum (FBS). DMEM, opti-MEM and FBS were all purchased from Gibco (ThermoFisher Scientific), and antibiotics were purchased from GE (Hyclone).

[0065] Lipofectamine 3000 used for transfection was purchased from ThermoFisher Scientific.

[0066] The extract used to extract nuclear protein was purchased from Shanghai Beibo Biotechnology Co., Ltd., and the protein content was determined using the BCA protein quantification kit (Biyuntian Biotechnology Co., Ltd.).

[0067] Dynabeads TM M-270 streptavidin magnetic beads were purchased from ThermoFisher Scientific, catalog number 65305.

[0068] Mass spectrometry grade trypsin was purchased from Promega.

[0069] Mass spectrometry grade water and acetonitrile were purchased from ThermoFisher Scientific.

[0070] Deionized water was prepared by Millipore water purification instrument.

[0071] The recombinant pcDNA3.1(+) plasmid was synthesized by Nanjing GenScript Co., Ltd., and the sequence is shown in SEQ ID NO: 5, wherein the coding sequence of the nuclear localization signal sequence is shown in nucleotides 1 to 21; the coding sequence of the recognition element BRD2 that recognizes histone modification is shown in nucleotides 43 to 609; the coding sequence of the loose sequence A is shown in nucleotides 1198 to 1393; the coding sequence of the biotin ligase BASU is shown in nucleotides 1405 to 2184; the coding sequence of the loose sequence B is shown in nucleotides 2185 to 2220; and the coding sequence of the green fluorescent protein is shown in nucleotides 2251 to 2970.

[0072] Example 1

[0073] This example is used to illustrate the amplification and extraction of probes.

[0074] Mix the probe plasmid with 100 μL DH5α cells, place on ice for 30 minutes, place in a 42°C water bath for 90 seconds, and place on ice for 2 minutes. Add the competent cells to the LB solid medium containing ampicillin, spread evenly, and after 16 hours of culture, pick a single colony on the medium and culture it in 900 mL of LB liquid medium containing ampicillin. After 16 hours, centrifuge and collect the bacteria to extract the plasmid. The extracted plasmid is about 1500 μg.

[0075] Example 2

[0076] This example is used to illustrate the transfection of the probe into A549 cells.

[0077] When the cell density reaches 50-80%, replace the culture medium with serum-free Opti-MEM medium. Take two centrifuge tubes, take 40 μg of plasmid in Opti-MEM medium in one tube, and add Lipofectamine at a ratio of 1:2 (m / v). TM 3000 reagent; add an appropriate amount of lipo3000 liposomes and Opti-MEM medium to another tube. Then mix the solutions in the two tubes, let stand for 20 minutes after fully mixing, and then add the solution dropwise to the medium. After 6 hours, change to normal serum medium for culture. After 24 hours of culture, change the medium to phenol red-free medium and observe the EGFP signal under a laser confocal microscope. The results are as follows Figure 2As shown, it can be observed that the green fluorescence signal is mainly concentrated in the cell nucleus.

[0078] Example 3

[0079] This example is used to illustrate the use of streptavidin magnetic beads to capture biotinylated proteins in nuclear proteins.

[0080] (1) Tumor cell culture and nuclear protein extraction

[0081] Human non-small cell lung cancer cells (A549) were cultured in DMEM medium containing 10% fetal bovine serum, 100U / mL penicillin, and 100mg / mL streptomycin. The probe plasmid was transfected at a plasmid dosage of 40μg per 1 million cells. The cells were then cultured in a medium containing 55μM biotin. After 24 hours, the cells were collected with a scraper, washed three times with PBS, and then 200μL of cold extract A was added. The mixture was vortexed and shaken on ice for 30min. Then, the mixture was centrifuged at 4°C, 2000g for 5min. The supernatant was discarded, and the cold extract B was added to the precipitate. The mixture was vortexed and shaken on ice for 40min. The supernatant was the nuclear protein extract, and the nuclear protein content was determined using a BCA kit.

[0082] (2) Capture of biotinylated proteins with streptavidin magnetic beads

[0083] 2 mg of the nuclear protein prepared in (1) was incubated with 1 mg of streptavidin magnetic beads (purchased from Thermo Fisher Scientific, catalog number 65305) for 16 h to capture the biotinylated protein. After incubation at 4°C for 16 h, the beads were separated on a magnetic rack and the supernatant was removed; the beads were washed with 0.1% PBST solution by volume, separated on a magnetic rack, the supernatant was removed (recorded as the flow-through solution), and the washing was repeated 4 times; the biotinylated protein bound to the magnetic beads was eluted with 100 μL of 0.1 mol / L glycine solution.

[0084] Figure 3 The results of Coomassie brilliant blue staining of the loading, elution and flow-through, and immunoblotting of streptavidin coupled to horseradish peroxidase, respectively, show that the amount of protein decreases after magnetic bead enrichment, but the proportion of biotinylated protein is higher, indicating that streptavidin magnetic beads can enrich biotinylated nuclear proteins in nuclear proteins.

[0085] Example 4

[0086] This example is used to illustrate the use of negative probes to exclude non-specific binding of cells and interference from self-biotinylated proteins in cells. The cells used are A549 cells.

[0087] (1) Capture of biotinylated proteins

[0088] According to the method of Example 3, a biotin-labeled probe and a negative probe without BRD2 were transfected into the cells respectively, and biotin was added to the cell culture medium 36 hours later. Nuclear protein was extracted 24 hours later and quantified by the BCA method. Biotinylated nuclear protein was captured by streptavidin, and the captured biotinylated protein was eluted with 0.1 mol / L glycine and spin-dried.

[0089] (2) Protein sample pretreatment

[0090] The captured protein was denatured with 8mol / L urea for 15min, tri(2-carboxyethyl)phosphine (TCEP, final concentration 5mmol / L) was added at 37℃ for 1h to reduce the disulfide bonds in the protein to free sulfhydryl groups, and then iodoacetamide (IAA, final concentration 10mmol / L) was added to react at room temperature in the dark for 45min to block the free sulfhydryl groups. After that, a 10K ultrafiltration tube was used to desalt and replace the buffer with 50mmol / L triethylammonium bicarbonate (TEAB), and then trypsin was added at a ratio of 1:50 (w / w), where 1 was trypsin and 50 was the enzymatically hydrolyzed protein, and the enzymatic hydrolysis was carried out at 37℃ for 16h. The peptides produced by the enzymatic hydrolysis were re-dissolved in 5μL 0.1% formic acid aqueous solution and then tested by nano-LC-MS.

[0091] (3) Nanoliter LC-MS testing

[0092] The nanoliter liquid phase used was EASY-nLC 1200 system (ThermoFisher Scientific). The chromatographic columns were C18 capture column (75 μm × 2 cm, 3 μm, ThermoFisher Scientific) and C18 analytical column (75 μm × 15 cm, 2 μm, ThermoFisher Scientific), and the mobile phases were water containing 0.1% formic acid (phase A) and acetonitrile / water containing 0.1% formic acid (80 / 20, v / v, phase B). A C18 analytical column was used for peptide separation, with a gradient of 0-15min 1% B-5% B, 15-85min 5% B-20% B, 85-110min 20% B-35% B, 110-117min 35% B-95% B, 117-121min 95% B, 121-123min 95% B-50% B, 123-127min 50% B, 127-130min 50% B-2% B, and a flow rate of 300nL / min. The nanoliter liquid phase was coupled to a mass spectrometer equipped with an orbital trap (Orbitrap Fusion Lumos Tribrid). After the peptides were separated by the C18 column, they entered the mass spectrometer for detection. The mass spectrometry acquisition mode was data-dependent mode, the mass-to-charge ratio range of the mass spectrometer scan was 350-1800, and the signal intensity was selected to be stronger than 4.0×104 Ions with a charge of 2+, 3+, 4+ or 5+ were subjected to HCD secondary fragmentation. The mass-to-charge ratio range of the secondary mass spectrometry acquisition was 110-2000, and the dynamic peak exclusion time was set to 30 s.

[0093] (4) Protein identification and relative quantitative comparison

[0094] Proteome Discoverer 2.4 software was used for mass spectrometry data analysis, peak extraction, protein sequence database search, protein identification and protein relative quantitative analysis. The Swiss-Prot (Human) database was used for database search, the maximum trypsin missed cleavage site was 3, the parent ion mass error was 10 ppm, the daughter ion mass error was 0.02 Da, and carbamidomethylation of cysteine ​​was set as a fixed modification; oxidation of methionine, phosphorylation of serine, threonine, and tyrosine, methylation of lysine, glutamine, and arginine, acetylation of lysine and arginine, and biotinylation of lysine were set as variable modifications. The mass spectrometry quantitative method is based on the ratio of the relative intensity of the extracted ion current chromatogram of the parent ion. The quantitative ratio of the protein is obtained by the weighted average of the ratio of peptides from the same protein in the sample corresponding to the positive probe and the sample corresponding to the negative probe. Proteins with relative quantitative p values ​​< 0.05 and quantitative ratios ≥ 1.20 or ≤ 0.83 are considered to be proteins that respond to cisplatin-damaged DNA.

[0095] Results: Only 10 proteins were identified in the group without recognition element BRD2, while 97 proteins were identified in the group with BRD2 ( Figure 4 ), indicating that the biotinylated probe with recognition element and the method of capturing and identifying proteins using the probe are specific, less interfered by the self-biotinylated protein in the cell, and can be used to capture and identify drug injury response proteins.

[0096] Example 5

[0097] This example is used to illustrate the use of capture and identification of cisplatin-damaged DNA response proteins, such as Figure 1 shown.

[0098] The biotin-labeled probe was transfected according to the method of Example 3. After 36 hours, a culture medium containing cisplatin and biotin was added for culture. The cisplatin concentration was 10 μM. The control group was a culture medium containing the same concentration of biotin but without cisplatin. After 24 hours, the cells were collected.

[0099] The nuclear protein collection, biotinylated protein enrichment and LC-MS detection methods are the same as those in Example 4.

[0100] The results of mass spectrometry are shown in Figure 5In the above analysis, the non-specific binding proteins identified in Example 4 were excluded, and finally 387 proteins were identified, and the number of enriched proteins was large. The GO analysis of the identified proteins showed that Figure 6 . Figure 6 The results showed that the identified proteins were mainly located in the cell nucleus (289 proteins, 74.7%), and their biological functions were mainly related to nucleic acid binding (265 proteins, 68.5%), indicating that the enriched proteins were mainly DNA binding proteins with good specificity.

[0101] Among the identified proteins, 38 were significantly decreased, that is, proteins with reduced DNA binding after cisplatin treatment, which are called negative response proteins. GO analysis enrichment of negative response proteins is shown in Figure 7 . The results showed that the down-regulated proteins were mainly related to RNA shearing, indicating that cisplatin affects gene transcription, which has also been reported in many literatures. In addition, 177 proteins were up-regulated, and their binding to DNA increased after cisplatin treatment. These proteins may be related to the recognition and repair of cisplatin DNA damage. In addition, the inventors of the present invention found that YB1, KU80, DDX5, etc., which were previously identified in the literature as binding to cisplatin-damaged DNA, were among the up-regulated proteins found, among which XRCC5 (KU80), NPM1, etc. were related to the recognition and repair of DNA damage caused by cisplatin, further illustrating that the probe provided by the present invention and the method of the present invention were used to capture the response proteins to DNA drug damage; Figure 8 The WB method was used to verify the changing trends of KU80 and NPM1, verifying the reliability of the mass spectrometry results, and indicating that the identified positive response proteins were involved in the recognition and repair of cisplatin DNA binding.

[0102] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

[0103] sequence:

[0104]

[0105]

Claims

1. A probe for capturing a protein responsive to drug-damaged DNA, characterized in that: The probe includes the following elements connected in tandem: (nuclear localization signal sequence-BRD2) n -Biotin ligase, wherein n is an integer of 1-4.

2. The probe according to claim 1, wherein The nuclear localization signal sequence is selected from at least one of a single type nuclear localization signal, a bipartite nuclear localization signal and a proline tyrosine nuclear localization signal, more preferably a single type nuclear localization signal; Further preferably, the amino acid sequence of the nuclear localization signal sequence is as shown in SEQ ID NO: 1; Preferably, n is an integer of 2-4; Preferably, the BRD2 is connected to the biotin ligase via a loose sequence A; More preferably, the loose sequence A consists of 10-13 polypeptide fragments GGGGS; Further preferably, the amino acid sequence of the loose sequence A is as shown in SEQ ID NO: 3; Preferably, the probe further comprises a marker protein; More preferably, the marker protein is a fluorescent protein; Preferably, the biotin ligase is at least one selected from BASU, BioID, BioID2, TurboID and BirA.

3. The probe according to claim 2, wherein: The labeled protein is linked to a biotin ligase; Preferably, the biotin ligase is connected to the marker protein via a loose sequence B; More preferably, the loose sequence B consists of 1-3 polypeptide fragments EAAAK; Further preferably, the amino acid sequence of the loose sequence B is as shown in SEQ ID NO:

4.

4. A method for preparing the probe according to any one of claims 1 to 3, characterized in that: The method involves introducing a plasmid expressing the probe into the target cell.

5. Use of the probe according to any one of claims 1 to 3 or the probe prepared by the method according to claim 4 in capturing and identifying proteins responsive to drug-damaged DNA.

6. A method for capturing a protein responsive to drug-damaged DNA, characterized in that: The method comprises the following steps: (1) introducing a plasmid expressing the probe according to any one of claims 1 to 3 into a target cell, and then contacting the target cell with biotin and a drug so that the biotin binds to the probe; (2) extracting nuclear proteins from target cells, separating captured biotinylated proteins, and then performing mass spectrometry detection; (3) Quantitative analysis of the identified proteins was performed to exclude nonspecifically bound proteins and intracellular self-biotinylated proteins, and to screen out proteins that respond to drug-damaged DNA.

7. The method according to claim 6, wherein: The quantitative analysis method includes using cells that have not been exposed to the drug as a control, excluding proteins that bind to undamaged DNA, and obtaining captured proteins that respond to drug-damaged DNA; The captured proteins that respond to drug-damaged DNA are identified by mass spectrometry-based quantitative proteomics, and the relative quantitative ratios of the proteins that respond to drug-damaged DNA to the proteins that bind to undamaged DNA are obtained. The proteins with significantly different abundances are proteins that specifically respond to drug-damaged DNA, and the identified proteins that specifically respond to drug-damaged DNA are verified by biochemical and molecular biological methods.

8. The method according to claim 7, wherein: Proteins with relative quantitative p values ​​< 0.05 and relative quantitative ratios ≥ 1.20 or ≤ 0.83 were considered to be specific response proteins to drug-damaged DNA.

9. The method according to any one of claims 6 to 8, wherein: In step (1), the number is 1×10 6 cells, the amount of the plasmid is 20-50 μg, preferably 35-45 μg; Preferably, relative to 40 μg of plasmid, the amount of biotin used is 0.5-100 μM; the contact time is 12-36 h, preferably 16-24 h.

10. The method according to any one of claims 6 to 8, wherein: The separation comprises: extracting the nucleoprotein of the target cell, combining the nucleoprotein with magnetic beads that specifically bind to the biotinylated protein, and then separating the nucleoprotein bound to the magnetic beads by attracting an external magnetic field.

Images