Method for detecting cysteine sulfinylation modification of target protein and application thereof

Abstract

The invention discloses a method for detecting cysteine sulfinylation modification of a target protein and application of the method. The method mainly comprises the following steps: S1, labeling cysteine sulfinylation modification sites of the target protein by using a BTD-Az probe; s2, connecting a molecular tag to the BTD-Az probe by adopting a copper-free click chemical reaction to capture a target protein; s3, extracting a target protein, enriching the target protein with a tag, and detecting the target protein; or extracting the target protein, enriching the target protein with the tag, removing the tag, and detecting the target protein. According to the method, the protein subjected to sulfinylation modification can be sensitively labeled with a molecular tag in situ in a living cell, the labeling process is simplified, in addition, the detection of the sulfinylation modification level can be carried out in an animal model, the dynamic level change of the protein sulfinylation modification can be detected in a state closer to a living body, and the detection accuracy is improved. Therefore, research on protein sulfinylation modification is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of protein or polypeptide post-translational modification detection, and specifically relates to a method for detecting cysteine ​​sulfenylation modification of a target protein and an application thereof. Background Art

[0002] The production of reactive oxygen species (ROS) is accompanied by the normal physiological or pathological processes of cells. When the body is damaged or diseased, high concentrations of ROS are produced, causing oxidative stress or oxidative post-translational modifications (OxiPTMs) of proteins. This process oxidizes the thiol group (-SH) of cysteine ​​(Cys) residues in proteins into sulfenylation (S-sulfenylation, -SOH), sulfinylation (-SO2H) or sulfonylation (S-sulfonylation, -SO3H) and other forms, causing oxidative damage to cells and tissues, and inducing diseases such as cancer, hypertension, Parkinson's disease or Alzheimer's disease. Among them, sulfenylation modification is a reversible, redox-dependent modification that can change the conformation and function of proteins, thereby affecting cell signal transduction, metabolism, growth and differentiation processes. Therefore, studying protein sulfenylation modification can provide a more comprehensive understanding of the survival mechanisms of various organisms, promote the understanding of the mechanisms of disease occurrence, and provide new targets for the diagnosis and treatment of diseases.

[0003] The capture, enrichment and determination of modification sites of sulfenylated proteins are still extremely challenging. The detection methods of cysteine ​​sulfenylation mainly include molecular probes based on the transcription factor Yap1 and small molecule compounds dimedone or bicyclo[6.1.0]nonyne. On this basis, more derivative probes that are convenient for enriching sulfenylated proteins have been designed by coupling label molecules such as biotin. After the sulfenylated proteins are captured and enriched, they can be combined with mass spectrometry analysis techniques such as LC-MS / MS to determine the cysteine ​​sites where sulfenylated modification occurs. For example, a BCN probe is used, or a BCN derivative is combined with a label dimethylpiperidine that is convenient for antibody enrichment for selective enrichment. The activated carbon between the two carbonyl groups in the dimedone-based detection probe is nucleophilic and easily attacks the hydroxyl group in the electrophilic Cys-SOH, and forms a covalent complex after removing one molecule of H2O. Currently, there are DCP and DYn-2 probes based on dimedone. The benzothiazine-based probe BTD can selectively label the S-sulfinic acid of protein cysteine. The alkyne group in the probe can be linked to photocleavable azido-biotin via CuAAC click chemistry to achieve the selective enrichment of sulfenylated cysteine. However, protein degradation may occur when biotin is linked via CuAAC click chemistry after cell lysis, and the activity of the protein is affected to a certain extent by the involvement of copper ions.

[0004] In summary, existing detection methods still have problems such as complicated procedures, time-consuming operations, inability to directly biotinylate sulfonylated proteins, and inability to label in animals. Summary of the invention

[0005] In view of this, the primary purpose of the present invention is to provide a method for detecting cysteine ​​sulfenylation of a target protein. The method uses a specific BTD probe combined with a copper-free click chemistry to connect a molecular tag, which can sensitively label the sulfenyl-modified protein in situ in living cells with a molecular tag, which not only simplifies the labeling process, but also can detect the sulfenylation modification level in an animal model, and detect the dynamic level changes of protein sulfenylation modification in a state closer to the living body.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for detecting cysteine ​​sulfenylation modification of a target protein. In the present invention, a specific BTD probe is combined with a copper-free click chemistry to connect a molecular tag, thereby achieving in situ labeling of a protein that undergoes sulfenylation modification with a molecular tag in a living cell, simplifying the labeling process, and detecting the dynamic level changes of protein sulfenylation modification in a state closer to the living body. The method of the present invention can not only detect proteins that undergo sulfenylation modification; it can also detect the modified amino acid sites of proteins that undergo sulfenylation modification.

[0008] In the present invention, the method for detecting cysteine ​​sulfenylation modification of a target protein mainly comprises the following steps:

[0009] S1. Use BTD-Az probe to label the target protein cysteine Sulfenyl acid Modification site

[0010] The molecular structure of the BTD-Az probe described in the present invention is as follows:

[0011]

[0012] The synthesis path is as follows:

[0013]

[0014] In specific operations, the prepared cells are cultured in a culture medium containing the BTD-Az probe to achieve labeling. The specific amount of the probe can be selected according to the number of cells, etc., and those skilled in the art have such ability, so there is no special limitation.

[0015] S2, using copper-free click chemistry reaction in the BTD-Az Probe connection Molecular tag capture of target protein

[0016] The copper-free click chemistry reaction used in the present invention is azide-alkyne cycloaddition (SPAAC). The copper-free click chemistry reaction includes the steps of connecting the BTD probe-labeled target protein with the biomolecule with the molecular tag. The specific connection method and reaction conditions can be confirmed by those skilled in the art based on different biomolecules by known methods, and will not be described in detail here.

[0017] In the present invention, the molecular tag is a common one in the art such as biotin, fluorescein (e.g., FAM), etc., but is not limited thereto. Depending on the different molecular tags used, the selected biomolecules with molecular tags will also be different. In some preferred embodiments of the present invention, the molecular tag used is biotin, and the biomolecules with molecular tags used are biomolecules with biotin tags. Specific examples that can be mentioned include PC DBCO-PEG3-Biotin, Diazo Biotin-PEG3-DBCO, Dde Biotin-PEG4-DBCO, UV Cleavable Biotin-PEG3-DBCO or DBCO-PEG4-Biotin, but are not limited thereto.

[0018] S3, extract the target protein and enrich the target protein with label, and detect the target protein; or, extract Target protein and enrich the target protein with label, remove the label, and detect the target protein

[0019] In some embodiments of the present invention, step S3 is to extract the target protein and enrich the target protein with the label, and then detect the target protein. After extracting the target protein and enriching the target protein with the label, the target protein is detected, thereby achieving the purpose of detecting the sulfenylation-modified protein.

[0020] In other embodiments of the present invention, step S3 is to extract the target protein and enrich the target protein with the tag, remove the tag, and detect the target protein. After extracting the target protein and enriching the target protein with the tag, removing the tag, and detecting the target protein, the purpose of detecting the modified amino acid site of the sulfenylation-modified protein is achieved.

[0021] Further, in the present invention, the protein enrichment method can adopt conventional methods in the art, which vary according to the different molecular tags selected. For example, in some implementation cases, the molecular tag is a biotin tag, and then streptavidin-biotin is used to achieve protein enrichment, wherein the form of streptavidin can be, for example, streptavidin magnetic beads or streptavidin gel. For example, in other implementation cases, the molecular tag is fluorescein (such as FAM), and then anti-FAM antibodies are used to achieve protein enrichment.

[0022] Similarly, there is no particular limitation on the method of removing the label, and the specific selection is made according to the different types of biological molecules with molecular labels. For example, in some specific implementation cases of the present invention, the biological molecules with molecular labels used are PC DBCO-PEG3-Biotin, and the subsequent label removal is achieved by photolysis release. For example, in some specific implementation cases of the present invention, the biological molecules with molecular labels used are Diazo Biotin-PEG3-DBCO, wherein the diazo group can be released under mild conditions of 50mM sodium dithionite solution. The specific removal method can be determined by those skilled in the art based on known types and methods of biological molecules.

[0023] Furthermore, in the present invention, the step of enriching the target protein with a label and removing the label can be achieved in a variety of ways:

[0024] For example, in some implementation cases, the purified target protein is digested, the target protein is enriched, and the tag is removed;

[0025] For example, in other implementation cases, the purified target protein is enriched, the tag is removed, and then digested;

[0026] For example, in other implementation cases, the purified target protein is enriched, and then the enriched target protein is digested and the tag is removed.

[0027] The above three methods can all achieve the purpose of enriching proteins and removing labels.

[0028] Among them, digestion is a routine operation in the art, and is generally performed by selecting appropriate digestion solutions and digestion conditions according to the type of target protein or peptide segment. Those skilled in the art have such capabilities, so there is no particular limitation. In some specific embodiments of the present invention, the digestion solution used is pancreatic enzyme, but it is not limited thereto.

[0029] In addition, it is understood that the method of extracting the target protein described in the present invention is a conventional operation in the art, for example, it can be achieved by lysing cells with a lysis buffer and then centrifuging, which will not be elaborated here.

[0030] The purification and enrichment of proteins can be carried out by conventional operations in the art without any special limitation. Usually, suitable purification and enrichment methods are selected according to the type of protein and the type of molecular tag.

[0031] Furthermore, the detection of the target protein can be performed using conventional methods in the art, such as LC-MS / MS, and the specific detection conditions can be based on the liquid chromatography conditions in the currently known common protein spectrum analysis.

[0032] Furthermore, the detection of the target protein in the present invention also includes the step of quantifying the cysteine ​​sulfenylation modification of the target protein, and the specific quantitative methods used include but are not limited to LFQ, DDA, DIA, probe isotope labeling, TMT, IBAQ or SILAC.

[0033] The second aspect of the present invention provides a kit for detecting cysteine ​​sulfenylation modification of a target protein, wherein the kit contains a BTD-Az probe and performs the method described in the first aspect of the present invention.

[0034] The kit described in the present invention refers to an in vitro product for non-therapeutic or diagnostic purposes. It is understood that in addition to the BTD-Az probe, the kit also contains other reagents or adjuvants, such as lysate, buffer, cleaning solution, digestion solution, etc., which vary depending on the steps used in the method and the detection method. There is no specific limitation here, and those skilled in the art can determine or configure according to known methods or means.

[0035] In addition, it should be noted that the target protein described in this article is not limited to proteins, but can also be enzymes or polypeptides.

[0036] Beneficial effects of the present invention:

[0037] The present invention uses the BTD-Az probe in combination with SPAAC copper-free click chemistry to sensitively label the sulfenyl-modified proteins in situ in living cells with molecular tags, further enrich and purify the modified protein peptides, and quantitatively detect the modification sites and modification level changes. Compared with the prior art, the method of the present invention has the following advantages:

[0038] (1) Compared with the traditional DYn-2 probe, the BTD-Az probe used in the present invention has a stronger binding ability to sulfenylation modification, shortens the time required for labeling, and has a stronger ability to capture dynamically occurring sulfenylation modification.

[0039] (2) Compared with the two-step scheme of first labeling the probe intracellularly, lysing the cells, and then using CuAAC to connect biotin, the method of the present invention avoids the protein degradation that may occur during CuAAC after cell lysis, as well as the influence of copper ions required for the reaction on protein activity, thereby improving the accuracy of detection.

[0040] (3) Compared with the requirements of the CuAAC reaction on the solution environment in the two-step scheme, and therefore the need for multiple strong cation exchange and other protein purification methods, the present invention uses SPAAC copper-free click chemistry, which can react in the cell physiological environment without the need for additional treatment. In the magnetic bead cleaning step, the detergent residue can be removed by multiple washings to avoid its influence on mass spectrometry detection, which greatly simplifies the processing flow and shortens the processing time.

[0041] In addition, the probes and methods used in the present invention can be used to label proteins that have undergone sulfenylation modification in the whole body or locally of living animals, and then the animals are killed and samples are collected for detection, thereby being able to detect changes in protein sulfenylation modification levels in a state closer to that of living organisms, which is more practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the process for detecting protein sulfenylation modification in Example 1.

[0043] Figure 2 This is a Venn diagram of proteins retrieved for the three replicates of the control group in Example 1.

[0044] Figure 3 Venn diagram of proteins retrieved for the three replicates of the RSL3 treatment group in Example 1.

[0045] Figure 4 This is a volcano plot of the differential analysis of proteins quantitatively detected between the control group and the RSL3-treated group in Example 1.

[0046] Figure 5 Schematic diagram of the process for detecting protein sulfenylation modification in Example 2.

[0047] Figure 6 The GO enrichment analysis results of 1446 proteins in Example 2.

[0048] Figure 7 The following are the results of KEGG enrichment analysis of 1446 proteins in Example 2.

[0049] Figure 8 The results of Western blotting for detecting sulfenylation modification of articular cartilage protein in animals in Example 3, wherein: Figure 8 A is the immunoblot band diagram of different BTD-Az labeling times. Figure 8 B is a statistical graph of the signal intensity of sulfenylation-modified proteins detected at different BTD-Az labeling times. DETAILED DESCRIPTION

[0050] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, unless otherwise specified, methods that do not specifically record conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0052] Example 1

[0053] This example discloses a method for detecting protein sulfenylation modification, the process is as follows Figure 1 As shown in , the specific steps are as follows:

[0054] S1. Synthesis of BTD-Az probe

[0055] In this step, the BTD-Az probe was synthesized according to the following reaction pathway:

[0056]

[0057] Step a: Dissolve the raw material A (5.00 g, 1.0 equiv) in 10 mL of DCM, add triethylamine (3.67 g, 1.2 equiv), cool to 0°C in an ice bath, slowly dropwise add methanesulfonyl chloride (4.16 g, 1.2 equiv), and react at room temperature for 3 hours under N2 protection. After the reaction is completed, add ice water to quench, extract, rinse with saturated brine, dry over anhydrous sodium sulfate, spin dry, and purify by column chromatography (PE / EA=2:1) ​​to obtain 6.51 g (yield 83.8%) of light yellow crystalline solid. [M+H+]=244.1, [M-H+]=242.1.

[0058] Step b: 1-Chloro-4-iodobutane (2.00 g, 1.5 equiv) was dissolved in 10 mL of anhydrous DMF, K2CO3 (1.68 g, 2.0 equiv) was added, and material B (1.48 g, 1.0 eq) was added after 15 minutes, and the mixture was reacted at 75°C for 5 hours under N2 protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with EA, washed with saturated brine, dried over anhydrous sodium sulfate, spin-dried, and purified by column chromatography to obtain 1.27 g (yield 62.6%) of a transparent liquid. [M+H+] = 334.1.

[0059] Step c: Dissolve NaH (0.275 g, 3 equiv) in 10 mL of anhydrous DMF, cool to 0°C in an ice bath, slowly dropwise add DMF solution (8 mL) containing material C (1.27 g, 1.0 equiv), and react at room temperature for 7 hours under N2 protection. After the reaction is completed, adjust the reaction solution to pH = 3 with 2N HCl, extract with EA, rinse with saturated brine, dry with anhydrous sodium sulfate, spin dry, and purify with column chromatography (PE / EA = 6:1) to obtain 0.43 g (yield 40.1%) of light green liquid. [M-H+] = 286.1.

[0060] Step d: Dissolve material D (0.43 g, 1.0 equiv) in 10 mL of anhydrous DMF, add NaN3 (0.19 g, 2.0 equiv), raise the reaction temperature to 50°C, and react for 6 hours. After the reaction is completed, adjust the reaction solution to pH = 9 with 2N NaOH solution, extract with EA, rinse with saturated brine, dry with anhydrous sodium sulfate, spin dry, and purify with column chromatography to obtain 0.14 g (yield 32.6%) of light green oily liquid. [M-H+] = 293.1.

[0061] 1H NMR(500MHz, CDCl3)δ8.15(d,J=7.9Hz,1H),7.67(t,J=7.9Hz,1H),7.21(m,J=27.3,8.2Hz,2H),4.31(s,2H),4.04–3.97 (m,2H),3.60(t,J=6.1Hz,1H),3.37(t,J=6.6Hz,1H),1.96(m,J=7.6Hz,1H)1.89(m,J=7.6Hz,2H),1.71(m,J=7.6Hz,1H).

[0062] S2. Cell preparation

[0063] In a 10 cm cell culture dish, 293T cells were cultured in DMEM medium containing 10% fetal bovine serum until the cell confluence was 70% to 90%, and about 1×10 7 Adherent cells or suspension cells. (1×10 7 A cardinal number of cells)

[0064] In this example, the level of intracellular sulfenylation modification in the early stage of ferroptosis was detected. Based on the above steps, the culture medium containing 1 mM cell ferroptosis inducer RSL3 was replaced for 30 minutes. At the same time, a culture medium containing an equal amount of DMSO as RSL3 solvent was added for 30 minutes as a control group.

[0065] S3, BTD-Az labeling

[0066] Remove the culture medium, add 10 ml of culture medium containing 1 mM BTD-Az (add 200 μl of 50 mM BTD-Az working solution to 10 ml of culture medium) to each base number of cells, and incubate at 37°C for 1 hour. Remove the culture medium and rinse the culture dish twice with pre-cooled PBS. The 50 mM BTD-Az working solution is prepared by adding the BTD-Az probe synthesized in step (1) to dimethyl sulfoxide (DMSO).

[0067] S4, SPAAC copper-free click chemistry to connect biotin

[0068] Add 10 ml of DMEM medium containing 100 μM DBCO-PEG4-biotin (BCD-39, Xi'an Kangfuno Biotechnology Co., Ltd.) and 10% fetal bovine serum to each cell number and incubate at 37°C for 1 hour. Remove the medium and rinse the culture dish 3 times with pre-cooled PBS. Add 1 mL of pre-cooled PBS, scrape the adherent cells on ice with a cell scraper, centrifuge at 4°C and 200g for 5 minutes, and discard the supernatant. At this time, the cell pellet can be quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0069] The structural formula of the probe connected to biotin after SPAAC reaction is as follows:

[0070]

[0071] S5. Protein Extraction

[0072] Add 500 μL of lysis buffer to each cell number to lyse the cells. Ultrasonic lyse the cells at 20% output power for 10 seconds, repeat twice, and place on ice for 10 minutes. Centrifuge at 4°C and 20,000 g for 20 minutes, and pipette the supernatant into a new centrifuge tube. At this point, the supernatant can be quickly frozen in liquid nitrogen and stored in a -80°C refrigerator;

[0073] Among them, the formula of Lysis buffer is shown in the following table:

[0074]

[0075] S6, Biotin-Streptavidin Enrichment

[0076] Wash streptavidin magnetic beads (P2151, Biotech) with TBS three times in advance. For each base number of cells, take 100 μL and place it in a 1.5 mL centrifuge tube for later use. Wash each tube with TBS three times on the magnetic rack and resuspend the magnetic beads with the original volume of TBS. Add the resuspended streptavidin magnetic beads to the cell lysate and incubate at 4°C overnight or at room temperature for 2 hours. Place the centrifuge tube on the magnetic rack for separation for 1 minute, wash 5 times with 500 μL of washing buffer, and place it on the magnetic rack for separation for 1 minute after each wash, and discard the supernatant; wash at least 5 times with 500 μL of 25 mM ammonium bicarbonate solution;

[0077] The formula of washing buffer is shown in the following table:

[0078]

[0079] S7, trypsin digestion

[0080] After enrichment and washing, add tris(2-carboxyethyl)phosphine (TCEP) to the final concentration of 10mM, add iodoacetamide (IAM) to the final concentration of 30mM, and incubate at room temperature for 30min with rotation and in the dark. Add 10μg of sequencing-grade trypsin (Promega, V5113) to each cell, incubate at 37℃ for 14-16h, and then stand on the magnetic stand for 1min, and retain the supernatant.

[0081] S8, LC-MS

[0082] The supernatant was concentrated to dryness in a vacuum centrifuge and reconstituted with 60 μL of ultrapure water. After centrifugation at 20,000 g for 5 min, 50 μL was purified by C18 desalting column and dissolved with 0.1% formic acid and 40% acetonitrile. The supernatant was concentrated to dryness in a vacuum centrifuge again and reconstituted with 20 μL of 0.1% formic acid.

[0083] An appropriate amount of peptides was taken from each sample and chromatographically separated using the nanoliter flow rate Easy nLC 1200 chromatography system (ThermoScientific). The chromatographic conditions were as follows:

[0084] Buffer: Liquid A is a 0.1% formic acid aqueous solution, and liquid B is a mixed solution of 0.1% formic acid, acetonitrile and water (of which acetonitrile is 80%). The chromatographic column is balanced with 100% liquid A. The sample is injected into the Trap Column (100μm×20mm, 5μm, C18, Dr.Maisch GmbH) and then passes through the chromatographic analysis column (75μm×150mm, 3μm, C18, Dr.Maisch GmbH) for gradient separation at a flow rate of 300nl / min. The liquid phase separation gradient is shown in the following table:

[0085]

[0086]

[0087] After peptide separation, DIA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive PLUS mass spectrometer (Thermo Scientific). The analysis time was 120 min, detection mode: positive ion, parent ion scan range: 350-1500 m / z, primary mass spectrometry resolution: 70000@m / z 200, AGC target: 1e6, primary Maximum IT: 50 ms. The secondary mass spectrometry analysis of peptides was acquired according to the following method: after each full scan, the secondary mass spectra (MS2 scan) of the 20 most intense parent ions were triggered, the secondary mass spectrometry resolution: 17500@m / z 200, AGC target: 1e5, secondary Maximum IT: 110 ms, MS2 Activation Type: HCD, Isolation window: 1.6 m / z, Normalized collision energy: 27.

[0088] Results and Analysis

[0089] The test results in Example 1 were searched in the mass spectrometry database using mass spectrometry analysis software. The fasta database of the corresponding species was obtained from the Uniprot website and loaded into the software. Variable modifications were set: Carbamidomethyl (C), Oxidation (M), Acetyl (Protein N-term). LFQ quantitative analysis was used to compare the samples. Through the above analysis method, the read signals of unique peptides greater than 1 were screened, and the following results were obtained:

[0090] Figure 2 The Venn diagram of proteins retrieved from the control group in Example 1 for three repetitions is shown below. Figure 3The Venn diagram of proteins retrieved for the three replicates of the RSL3 treatment group; the LFQ intensity of the proteins quantitatively detected in the control group and the RSL3 treatment group was compared, and the difference analysis was performed with the LFQ intensity difference (i.e., fold change, FC) greater than 1.2 times as the threshold, and the volcano diagram was obtained ( Figure 4 ).

[0091] The above results indicate that the method in Example 1 can detect that in the early stage of ferroptosis induced by RSL3 treatment, the sulfenylation level of some proteins is increased.

[0092] Example 2

[0093] This example discloses a method for detecting sulfenylation-modified amino acid sites in proteins. The process is as follows: Figure 5 As shown in , the specific steps are as follows:

[0094] Steps S1 to S3 are implemented in the same manner as in Example 1.

[0095] S4, SPAAC copper-free click chemistry to connect biotin

[0096] 10 ml of culture medium containing 100 μM PC DBCO-PEG3-biotin (BCC-29, Xi'an Kangfuno Biotechnology Co., Ltd.) was added to each base number of cells, and the remaining steps were the same as step S4 in the aforementioned Example 1. The structural formula of the probe connected to biotin after the SPAAC reaction is as follows:

[0097]

[0098] S5. Protein Extraction

[0099] Same as Example 1.

[0100] S6. Trypsin digestion

[0101] Add TCEP to a final concentration of 10mM and IAM to a final concentration of 30mM to each base number of cells, and incubate at room temperature in the dark for 30min with rotation. Then add 500μL methanol and 125μL chloroform, and shake thoroughly to mix. Centrifuge at 1700g for 20min at 4℃ to discard the upper and lower liquid layers, retain the middle protein layer, and resuspend the precipitate with 500μL methanol / chloroform 1:1 solution, centrifuge at 16000g for 10min at 4℃ to discard the supernatant to wash the protein precipitate; repeat the wash once more and air-dry the protein precipitate.

[0102] The protein precipitate was dissolved in trypsin digestion buffer (about 500 μL) and sonicated;

[0103] Trypsin digestion buffer recipe:

[0104]

[0105] The concentration of BCA was measured and adjusted to 2 mg / mL. 10 μg of sequencing grade trypsin (Promega, V5113) was added at a ratio of trypsin / protein = 1:50 (wt / wt), incubated at 37°C for 14-16 h, centrifuged at 20000 g for 5 min, and the supernatant was retained.

[0106] S7, Biotin-Streptavidin Enrichment

[0107] Wash streptavidin magnetic beads (P2151, Biotech) with TBS three times in advance. For each base number of cells, take 100 μL and place it in a 1.5 mL centrifuge tube for later use. Wash each tube with TBS three times on the magnetic rack and resuspend the magnetic beads with the original volume of TBS. Add the resuspended streptavidin magnetic beads to the cell lysate and incubate at 4°C overnight or at room temperature for 2 hours. Place the centrifuge tube on the magnetic rack for separation for 1 minute, wash 5 times with 500 μL of washing buffer, and place it on the magnetic rack for separation for 1 minute after each wash, and discard the supernatant; wash at least 5 times with 500 μL of 25 mM ammonium bicarbonate solution;

[0108] The formula of washing buffer is shown in the following table:

[0109]

[0110]

[0111] S7, UV photolysis release

[0112] Add 600 μL (more than 6 times the volume of beads) of 25 mM ammonium bicarbonate solution and irradiate with 365 nm UV light for 30 min (5 mW / cm 2 ). Let stand on the magnetic rack for 1 min and keep the supernatant.

[0113] S8, LC-MS

[0114] Same as Example 1.

[0115] Results and Analysis

[0116] The same analysis method as in Example 1 was used to retrieve 1446 proteins, which were subjected to enrichment analysis. The results of GO enrichment and KEGG enrichment analysis were as follows: Figure 6 and Figure 7 shown.

[0117] pass Figure 6 and Figure 7The analysis results show that this method can identify peptides containing sulfenylation modifications in cells; through GO and KEGG enrichment results, it can be found that sulfenylation modifications mainly occur in the biological processes of translation, cytoplasm and RNA binding, and are closely related to pathways such as ribosomes, proteasomes and TCA cycle.

[0118] Example 3

[0119] This example discloses a method for detecting sulfenylation modification of articular cartilage protein in an experimental animal model. The specific steps are as follows:

[0120] S1. Reagent configuration

[0121] The BTD-Az powder synthesized in Example 1 was dissolved in DMSO to prepare a 100 mM storage solution, and diluted with PBS buffer to prepare a working solution with a final concentration of 1 mM. The DBCO-PEG4-Biotin powder in Example 1 was dissolved in DMSO to prepare a 10 mM storage solution, and diluted with PBS buffer to prepare a working solution with a final concentration of 100 μM.

[0122] S2. Mouse preparation

[0123] C57BL / 6 mice were taken and anesthetized with isoflurane gas. After anesthesia, the mice were fixed in a supine position, the hair around the knee joint was cleaned with a razor, and the skin around the knee joint was disinfected with alcohol.

[0124] S3, BTD-Az labeling

[0125] Flex the knee joint to fully expose the patellar tendon, puncture the outer edge of the patellar tendon below the patella, and insert the needle in the direction of the bisector of the angle of knee bending. There is a sense of emptiness when entering the joint cavity. Stop inserting the needle at this point, and inject 100μL of BTD-Az working solution with a 1mL syringe or 40unit insulin needle, and rotate to withdraw the needle.

[0126] S4. Joint lavage

[0127] At 1h, 2h and 4h after the injection of BTD-Az working solution, 50μL of PBS buffer was injected at the above injection site using the same technique, and the fluid in the joint cavity was aspirated; then 100μL of PBS buffer was injected, and the fluid in the joint cavity was aspirated.

[0128] At the same time, a control group was set up. The same technique was adopted in the control group. PBS buffer containing an equal amount of DMSO was injected into the joint cavity, and the joint cavity was lavaged using the same technique and process 4 hours after the injection.

[0129] S4. In vivo SPAAC

[0130] 100 μL of DBCO-PEG4-Biotin working solution was injected into the above injection site using the same technique.

[0131] S5. Sampling

[0132] One hour after SPAAC injection in vivo, mice were killed by cervical dislocation, the skin was incised in front of the patella, the joint capsule was incised on the medial side of the patella, the patella was everted to expose the joint cavity, the joint capsule and tendon ligaments inside and around the joint were cut, the knee joint was severed, and the tibial cartilage and femoral cartilage were cut with a scalpel.

[0133] Place the excised cartilage in pre-cooled PBS buffer, shake vigorously to clean the blood on the surface, take out the cartilage block and place it in a 2 mL grinding tube (which can be temporarily stored in an ultra-low temperature refrigerator).

[0134] S6. Protein Extraction

[0135] Precool the freezing grinder fixture in a deep freezer, add 5 3mm zirconia grinding beads to the 2mL containing cartilage blocks, put it into the freezing grinder (Shanghai Jingxin JXFSTPRP-24L), pour liquid nitrogen to assist in cooling, run at 50Hz for 30s, pause for 5s, and cycle 5 times. Add 200μL RIPA (strong) lysis buffer (Biyuntian P0013B), mix thoroughly, transfer the lysate to a new 1.5mL centrifuge tube, centrifuge at 15000g for 20min at 4℃, take the supernatant and perform BCA protein concentration detection according to the instructions of the kit (Biyuntian P0010). Dilute with PBS to adjust the protein concentration of each group to a consistent level, add an equal amount of 2×SDS loading buffer, and boil the sample for Western blot detection. Use streptavidin coupled to horseradish peroxidase to develop the bands, and the results are as follows. Figure 8 shown.

[0136] pass Figure 8 The results show that as the BTD-Az labeling time increases, the detected sulfenylation-modified protein signal intensity reaches the highest at 2 hours and decreases at 4 hours. This indicates that the method can be applied in animals and detected by conventional methods in the art such as Western blot, thereby realizing the detection of sulfenylation-modified proteins in animals.

[0137] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0138] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting cysteine ​​sulfenylation modification of a target protein, characterized in that: The following steps are involved: S1. Use BTD-Az probe to label the cysteine ​​sulfenylation modification site of the target protein; S2, using copper-free click chemistry to connect molecular tags to the BTD-Az probe to capture the target protein; S3, extracting the target protein and enriching the target protein with the label, and detecting the target protein; or, after extracting the target protein and enriching the target protein with the label, removing the label, and detecting the target protein; Wherein, the molecular structure of the BTD-Az probe is as follows:

2. The method according to claim 1, characterized in that The copper-free click chemistry reaction is SPAAC.

3. The method according to claim 1, characterized in that The copper-free click chemistry reaction includes the step of connecting a BTD probe-labeled target protein to a biological molecule with a molecular tag.

4. The method according to any one of claims 1 to 3, characterized in that: The molecular tag is biotin or fluorescein; Preferably, the molecular tag is biotin.

5. The method according to claim 4, characterized in that The biomolecule with a molecular tag is PC DBCO-PEG3-Biotin, Diazo Biotin-PEG3-DBCO, Dde Biotin-PEG4-DBCO, UV Cleavable Biotin-PEG3-DBCO or DBCO-PEG4-Biotin.

6. The method according to claim 1, characterized in that The process of extracting the target protein is achieved by lysing the cells with a lysis buffer followed by centrifugation.

7. The method according to claim 1, characterized in that The steps of enriching the target protein with a label and removing the label are as follows: After digesting the purified target protein, the target peptides are enriched and the tags are removed; Alternatively, after enriching the purified target protein, remove the tag and then digest it; Alternatively, the purified target protein is enriched, and then the enriched target protein is digested before removing the tag; Preferably, the enrichment is achieved by coupling streptavidin to biotin; Preferably, the streptavidin is in the form of streptavidin magnetic beads or streptavidin gel.

8. The method according to claim 1, characterized in that The detection was performed using LC-MS / MS.

9. The method according to claim 1, characterized in that The detection also includes the step of quantifying the cysteine ​​sulfenylation modification of the target protein; Preferably, the quantitative method adopts LFQ, DDA, DIA, probe isotope labeling, TMT, IBAQ or SILAC.

10. A kit for detecting cysteine ​​sulfenylation modification of a target protein, characterized in that: The kit contains a BTD-Az probe and performs the method according to any one of claims 1 to 9.

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