Detection kit for S-isopentene modified protein and use method of detection kit

Abstract

The invention discloses a detection kit for detecting S-isopentene modified protein, which comprises a first solution, a second solution, a third solution, a fourth solution and a fifth solution, the first solution is a first labeling reagent MAlk-FOH probe, the second solution is a lysis solution, the third solution is a second labeling reagent, the fourth solution is a sealing reagent, and the fifth solution is a second labeling reagent. The fifth solution is a horse radish peroxidase antibody. According to the invention, the MAlk-FOH probe for identifying the S-isopentenylation modification is used for culturing cells and marking the protein, so that the sensitive detection of the S-isopentenylation modified protein can be realized.

Description

Technical Field

[0001] This invention relates to testing or analyzing materials by measuring their chemical or physical properties, and specifically to a detection kit for detecting S-isoprenated modified proteins and its method of use. Background Technology

[0002] S-isopentenylated proteins are lipid modifiers that regulate membrane-protein and protein-protein interactions in cell signaling. Traditional methods use radioactive substrates for metabolic labeling, but these substrates have low sensitivity and require long detection times. Detection can be achieved by binding fluorescent isoprene diphosphates (NBD-GPP and NBD-FPP) and biotinylated (BGPP) analogs to isopentenylated proteins in mammalian cells. However, the size of the fluorophore or biotin moiety can interfere with protein membrane targeting and downstream signaling, resulting in significant interference and decreased accuracy. While isopentenylated proteins can be detected using isopentenylated probes (alk-FOH), they cannot distinguish between S-isopentenylated protein subtypes or proteins modified with geranyl or farnesyl groups, leading to insufficient accuracy. Therefore, developing a highly sensitive and easy-to-use detection method that can accurately distinguish modification subtypes is crucial.

[0003] Chinese invention patent application CN106062560A discloses a method for detecting protein modification using specific antibodies. It utilizes antibodies specific to modification sites linked to different dyes to detect the analyte, while simultaneously using antibodies specific to the unmodified analyte to determine its concentration. This method is used for quantifying modified analytes in the same sample, but it suffers from low sensitivity and long detection time. Chinese invention patent CN109072281B discloses an isopentenylation assay method. It provides a sample containing REP1; contacts the sample with Rab6a, Rab geraniylgeraniyltransferase, and a lipid donor substrate; and detects the lipidated Rab6a product to determine the activity of Rab escort protein 1. However, it cannot distinguish between isopentenylated protein subtypes. Summary of the Invention

[0004] To develop a detection method that can accurately distinguish modified subtypes, is highly sensitive, and is easy to operate, the first aspect of the present invention provides a detection kit comprising a first solution, a second solution, a third solution, a fourth solution, and a fifth solution, wherein the first solution is a first labeling reagent MAlk-FOH probe, the second solution is a lysis buffer, the third solution is a second labeling reagent, the fourth solution is a blocking reagent, and the fifth solution is a horseradish peroxidase antibody.

[0005] In one embodiment, the test kit further includes a sixth solution, which is a farnesyl inhibitor.

[0006] In one embodiment, the test kit further includes a seventh solution, which is a geraniol inhibitor.

[0007] In one embodiment, the MAlk-FOH probe is selectively oxidized at the terminal allyl group of farnesol to obtain a functionalized farnesol derivative; then, allyl alcohol is alkylated with propyne bromide and deprotected in acidic ethanol to generate Alk-FOH. The MAlk-FOH probe is stored at 4°C after preparation.

[0008] As one implementation, the working concentration of the MAlk-FOH probe is 50 µM.

[0009] Using the MAlk-FOH probe at a working concentration of 50 µM, S-farnylated and S-geraniolgeraniolized proteins can be sensitively detected by fluorescence using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) method. Higher concentrations result in cytotoxicity, while lower concentrations do not produce a highly sensitive and specific response.

[0010] In one embodiment, the pH value of the lysis solution is 7.5-8.5; the lysis solution includes urea, tris(hydroxymethyl)aminomethane, hydrochloric acid and double-distilled water.

[0011] As one embodiment, the method for preparing the lysis buffer includes adding 48.048g of urea, 302.85g of tris(hydroxymethyl)aminomethane, and 2.5mL of hydrochloric acid to double-distilled water to a final volume of 100mL to obtain a mixed solution with pH 8.0, which is then stored at 4°C for later use.

[0012] In one embodiment, the second labeling reagent comprises a DMSO solution of N-(3-azidopropyl)biotinamine, an aqueous solution of copper sulfate pentahydrate, an aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride, and a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.

[0013] In one embodiment, the preparation method of the second labeling reagent includes: 164 μg N-(3-azidopropyl)biotinamine, added to DMSO to 50 μL to obtain a DMSO solution of N-(3-azidopropyl)biotinamine; 1248 μg copper sulfate pentahydrate, added to water to 100 μL to obtain an aqueous solution of copper sulfate pentahydrate; 1430 μg tris(2-carboxyethyl)phosphine hydrochloride, added to water to 100 μL to obtain an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride; 265.3 μg tris[(1-benzyl-1H-1,2,3-triazol-4-yl)] [Methyl]amine was added to DMSO to 250 μL to obtain a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; a second labeling reagent was prepared by mixing a DMSO solution of N-(3-azidopropyl)biotinamine, an aqueous solution of copper sulfate pentahydrate, an aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride, and a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, and stored at 4 °C for later use.

[0014] In one embodiment, the blocking agent is 0.1M N-ethylmaleimide.

[0015] In one embodiment, the concentration of horseradish peroxidase (HRP) antibody in the fifth solution is 1 mg / mL.

[0016] As one embodiment, the farnesyl inhibitor is 10 mM N-[4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl]-(S)-methionine methyl ester hydrochloride ((S)-methyl 2-(5-(((R)-2-amino-3-mercaptopropyl)amino)-[1,1'-biphenyl]-2-ylcarboxamido)-4-(methylthio)butanoate hydrochloride).

[0017] In one embodiment, the geranyl inhibitor is 10 mM (S)2-(4-(((R)-2-amino-3-mercaptopropyl)amino)-2-(naphthalen-1-yl)benzamido)-4-methylpentanoate 2,2,2-trifluoroacetate.

[0018] A second aspect of the invention provides an application of the above-described detection kit, wherein the detection kit, which does not contain the sixth and seventh solutions, is used to detect S-isoprenated modified proteins.

[0019] A third aspect of the present invention provides an application of the above-described detection kit, wherein the detection kit containing the sixth solution is used to detect S-geranyl-modified proteins.

[0020] A fourth aspect of the present invention provides an application of the above-described detection kit, wherein the detection kit containing the seventh solution is used to detect S-farnesi modified proteins.

[0021] A fifth aspect of the present invention provides a method of using a kit for detecting S-isoprenated modified proteins, comprising the following steps: Add the first solution to the cell culture medium, culture the cells for 12 hours, wash the cells with PBS, and centrifuge to obtain the cell pellet; Add 190 μL of the second solution and 10 μL of 0.1 M benzyl sulfonyl fluoride (PMSF) to the obtained cell pellet to lyse the cells. Sonicate at 300 W for 5 seconds, pause for 5 seconds, continue for 2 min, centrifuge at 12000 g, 4 °C for 5 min, collect the supernatant, measure the concentration using the BCA method, take 500 μg of the supernatant into a 0.5 mL EP tube, and dilute with PBS to 180 μL to obtain the protein solution. Add 20 μL of the third solution to the obtained protein solution, invert at room temperature for 1-3 hours, then add 15 μL of the fourth solution, invert at room temperature for 1-5 hours to obtain the mixture; Add 600 μL of pre-cooled methanol, 150 μL of chloroform, and 400 μL of ddH2O to the obtained mixture, centrifuge at 4℃ and 6000 rpm for 15 min. A clearly visible milky white membrane is visible in the middle of the solution. Aspirate the upper and lower layers of liquid from the milky white membrane, add 600 μL of pre-cooled methanol, centrifuge at 4℃ and 6000 rpm for 5 min to remove the upper methanol layer, allow the precipitate to air dry naturally for 2-3 min, add 200 μL of the second solution, shake to mix and dissolve the precipitate, and determine the protein concentration to verify WB (WB) detection.

[0022] In one embodiment, 20 μL of a third solution is added to the obtained protein solution, and the mixture is inverted at room temperature for 1.5 h. Then, 15 μL of a fourth solution is added, and the mixture is inverted at room temperature for 4 h to obtain a mixture.

[0023] In one embodiment, 20 μL of the third solution is added to the obtained protein solution, inverted at room temperature for 1.5 h, then 15 μL of the fourth solution is added, and 2 μL of 0.1 M PMSF is added and inverted overnight at 4 °C to obtain a mixture.

[0024] As one implementation method, the method for detecting S-geranyl-modified proteins includes the following steps: Add the sixth solution (working concentration of 10 µM) to the cell culture medium and culture for 1-2 h, then add the first solution and culture for 12 h. Wash the cells and centrifuge to obtain cell pellet. Add 190 μL of the second solution and 10 μL of 0.1 M benzyl sulfonyl fluoride (PMSF) to the obtained cell pellet to lyse the cells. Sonicate at 300 W for 5 seconds, pause for 5 seconds, continue for 2 min, centrifuge at 12000 g, 4 °C for 5 min, collect the supernatant, measure the concentration using the BCA method, take 500 μg of the supernatant into a 0.5 mL EP tube, and dilute with PBS to 180 μL to obtain the protein solution. Add 20 μL of the third solution to the obtained protein solution, invert at room temperature for 1-3 hours, then add 15 μL of the fourth solution, invert at room temperature for 1-5 hours to obtain the mixture; Add 600 μL of pre-cooled methanol, 150 μL of chloroform, and 400 μL of ddH2O to the obtained mixture, centrifuge at 4℃ and 6000 rpm for 15 min. A clearly visible milky white membrane is visible in the middle of the solution. Aspirate the upper and lower layers of liquid from the milky white membrane, add 600 μL of pre-cooled methanol, centrifuge at 4℃ and 6000 rpm for 5 min to remove the upper methanol layer, allow the precipitate to air dry naturally for 2-3 min, add 200 μL of the second solution, shake to mix and dissolve the precipitate, and measure the protein concentration to verify Western blotting.

[0025] As one implementation method, the method for detecting S-farnesyl modified proteins includes the following steps: Add the seventh solution (working concentration of 5 µM) to the cell culture medium and culture for 1-2 h, then add the first solution and culture for 12 h. Wash the cells and centrifuge to obtain the cell pellet. Add 190 μL of the second solution and 10 μL of 0.1 M benzyl sulfonyl fluoride (PMSF) to the obtained cell pellet to lyse the cells. Sonicate at 300 W for 5 seconds, pause for 5 seconds, continue for 2 min, centrifuge at 12000 g, 4 °C for 5 min, collect the supernatant, measure the concentration using the BCA method, take 500 μg of the supernatant into a 0.5 mL EP tube, and dilute with PBS to 180 μL to obtain the protein solution. Add 20 μL of the third solution to the obtained protein solution, invert at room temperature for 1-3 hours, then add 15 μL of the fourth solution, invert at room temperature for 1-5 hours to obtain the mixture; Add 600 μL of pre-cooled methanol, 150 μL of chloroform, and 400 μL of ddH2O to the obtained mixture, centrifuge at 4℃ and 6000 rpm for 15 min. A clearly visible milky white membrane is visible in the middle of the solution. Aspirate the upper and lower layers of liquid from the milky white membrane, add 600 μL of pre-cooled methanol, centrifuge at 4℃ and 6000 rpm for 5 min to remove the upper methanol layer, allow the precipitate to air dry naturally for 2-3 min, add 200 μL of the second solution, shake to mix and dissolve the precipitate, and obtain the protein solution. Measure the protein concentration to verify the Western blot (WB).

[0026] As one implementation method, the method for verifying WB includes the following steps: The protein solution was separated by SDS-PAGE gel separation and the protein was transferred onto a PVDF membrane; Wash the protein-containing PVDF membrane twice, 10 minutes each time; Prepare a 3wt% BSA solution with 1XTBS-T and seal the cleaned PVDF membrane at room temperature for 1 hour. Clean the sealed PVDF membrane with 1XTBS-T for 10 min; Prepare a 3wt% BSA solution with 1XTBS-T, dilute with water to a volume ratio of 1:10000, and incubate the membrane overnight at 4°C. The PVDF membrane was cleaned three times with 1XTBS-T, each time for 10 minutes. Detection was performed using a chemiluminescence analyzer.

[0027] This invention utilizes the MAlk-FOH probe, which identifies S-isopentenylation modification, to culture cells, label proteins, and sensitively detect proteins with S-isopentenylation modification. Furthermore, it employs a geranyl inhibitor combined with a probe to culture cells, inhibiting geranyl modification formation and allowing the probe to label the farnesyl group, thus enabling the detection of proteins with farnesylation modification. Compared to existing technologies, this invention solves the problems of error-prone biochemical detection of protein S-isopentenylation, inability to distinguish between farnesyl and geranyl modifications, high difficulty, and long detection time.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) The detection kit for S-isopentenylated modified proteins described in this invention uses the MAlk-FOH probe that recognizes S-isopentenylation to culture cells and label the protein, which can achieve sensitive detection of S-isopentenylated modified proteins.

[0029] (2) The detection kit for S-isoprenylated modified proteins described in this invention uses geranyl inhibitor plus probe to culture cells, inhibits the formation of geranyl modification, and labels the probe on farnesyl to detect proteins that have undergone farnesylation modification.

[0030] (3) The detection kit for S-isoprenylated modified proteins described in this invention uses farnesyl inhibitors and probes to culture cells, inhibiting the formation of farnesyl modification, so that the probes are labeled on geranylide, and sensitively detect proteins that have undergone geranylide modification.

[0031] (4) The detection kit for S-isopentenyl modified proteins described in this invention solves the problems of easy error in the biochemical detection of S-isopentenyl protein, inability to distinguish between farnesyl modification and geranyl modification, high difficulty and long time consumption.

[0032] (5) The detection kit for S-isopentenylated modified proteins described in this invention uses a MAlk-FOH probe with a working concentration of 50 µM to perform sensitive fluorescence detection of S-farniated and S-geranylgeranylated proteins using a copper (I) catalyzed azide-alkyne cycloaddition method, while avoiding cytotoxicity. Attached Figure Description

[0033] Figure 1 The results are from the detection kit of Example 1. In the figure: M: protein molecular weight marker; 1 and 2 are parallel samples of Example 1.

[0034] Figure 2 The graph shows a comparison of the detection results of the detection kits in Examples 1 and 2. In the graph: M: Protein molecular weight marker; 3: Example 2; 4: Example 1.

[0035] Figure 3 The results of processing WB with ECL developing solution for the detection kit of Example 1 are shown.

[0036] Figure 4 The mass spectra of the classical isopentenyl modified protein identified by the detection kit correspond to the EITSSISC(1)PK protein.

[0037] Figure 5 The mass spectra of the classical isopentenyl modified protein identified by the detection kit correspond to the KEGLNGSSMC(1)SIMK protein.

[0038] Figure 6 The mass spectra of the classical isopentenyl modified protein identified by the detection kit correspond to the SLVELNHVC(1)K protein. Detailed Implementation

[0039] Example 1 A detection kit for S-isoprenylated modified proteins, the detection kit comprising a first solution, a second solution, a third solution, a fourth solution, and a fifth solution, wherein the first solution is a first labeling reagent, a MAlk-FOH probe; the second solution is a lysis buffer; the third solution is a second labeling reagent; the fourth solution is a blocking reagent; and the fifth solution is a horseradish peroxidase antibody.

[0040] The MAlk-FOH probe is prepared by selectively oxidizing the terminal allyl group of farnesol to obtain a functionalized farnesol derivative; then, allyl alcohol is alkylated with propyne bromide and deprotected in acidic ethanol to generate Alk-FOH. The MAlk-FOH probe is stored at 4°C after preparation.

[0041] The working concentration of the MAlk-FOH probe is 50 µM.

[0042] The preparation method of the lysis buffer includes adding 48.048g of urea, 302.85g of tris(hydroxymethyl)aminomethane, and 2.5mL of hydrochloric acid to double-distilled water to a final volume of 100mL to obtain a mixed solution with pH 8.0, which is then stored at 4°C for later use.

[0043] The preparation method of the second labeling reagent includes: 164 μg N-(3-azidopropyl)biotinamine, added to DMSO to 50 μL to obtain a DMSO solution of N-(3-azidopropyl)biotinamine; 1248 μg copper sulfate pentahydrate, added to water to 100 μL to obtain an aqueous solution of copper sulfate pentahydrate; 1430 μg tris(2-carboxyethyl)phosphine hydrochloride, added to water to 100 μL to obtain an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride; 265.3 μg tris[(1-benzyl-1H-1,2,3-triazol-4-yl] [Methyl]amine was added to DMSO to 250 μL to obtain a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; a second labeling reagent was prepared by mixing a DMSO solution of N-(3-azidopropyl)biotinamine, an aqueous solution of copper sulfate pentahydrate, an aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride, and a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, and stored at 4 °C for later use.

[0044] The blocking reagent is 0.1M N-ethylmaleimide.

[0045] The concentration of horseradish peroxidase antibody in the fifth solution is 1 mg / mL.

[0046] A method for using a detection kit for S-isoprenylated modified proteins includes the following steps: Add the first solution to the cell culture medium, culture the cells for 12 hours, wash the cells with PBS, and centrifuge to obtain the cell pellet; Add 190 μL of the second solution and 10 μL of 0.1 M benzyl sulfonyl fluoride to the obtained cell pellet to lyse the cells. Sonicate at 300 W for 5 seconds, pause for 5 seconds, continue for 2 min, centrifuge at 12000 g, 4 °C for 5 min, collect the supernatant, measure the concentration using the BCA method, take 500 μg of the supernatant into a 0.5 mL EP tube, and dilute with PBS to 180 μL to obtain the protein solution. Add 20 μL of the third solution to the obtained protein solution, invert at room temperature for 1.5 h, then add 15 μL of the fourth solution, invert at room temperature for 4 h to obtain the mixture; Add 600 μL of pre-cooled methanol, 150 μL of chloroform, and 400 μL of ddH2O to the obtained mixture, centrifuge at 4℃ and 6000 rpm for 15 min. A clearly visible milky white membrane is visible in the middle of the solution. Aspirate the upper and lower layers of liquid from the milky white membrane, add 600 μL of pre-cooled methanol, centrifuge at 4℃ and 6000 rpm for 5 min to remove the upper methanol layer, allow the precipitate to air dry naturally for 2-3 min, add 200 μL of the second solution, shake to mix and dissolve the precipitate, and obtain the protein solution. Measure the protein concentration to verify the Western blot (WB).

[0047] The method for verifying WB includes the following steps: The protein solution was separated by SDS-PAGE gel separation and the protein was transferred onto a PVDF membrane; Wash the protein-containing PVDF membrane twice, 10 minutes each time; Prepare a 3wt% BSA solution with 1XTBS-T and seal the cleaned PVDF membrane at room temperature for 1 hour. Clean the sealed PVDF membrane with 1XTBS-T for 10 min; Prepare a 3wt% BSA solution with 1XTBS-T, dilute with water to a volume ratio of 1:10000, and incubate the membrane overnight at 4°C. The PVDF membrane was cleaned three times with 1XTBS-T, each time for 10 minutes. Detection was performed using a chemiluminescence analyzer. Results are shown below. Figure 3 .

[0048] Example 2 A detection kit for S-isoprenylated modified proteins, the detection kit comprising a first solution, a second solution, a third solution, a fourth solution, a fifth solution, and a sixth solution, wherein the first solution is a first labeling reagent, a MAlk-FOH probe; the second solution is a lysis buffer; the third solution is a second labeling reagent; the fourth solution is a blocking reagent; the fifth solution is a horseradish peroxidase antibody; and the sixth solution is a farnesyl inhibitor.

[0049] The S-isoprenylated modified protein is an S-geranyl-modified protein.

[0050] The MAlk-FOH probe is prepared by selectively oxidizing the terminal allyl group of farnesol to obtain a functionalized farnesol derivative; then, allyl alcohol is alkylated with propyne bromide and deprotected in acidic ethanol to generate Alk-FOH. The MAlk-FOH probe is stored at 4°C after preparation.

[0051] The working concentration of the MAlk-FOH probe is 50 µM.

[0052] The preparation method of the lysis buffer includes adding 48.048g of urea, 302.85g of tris(hydroxymethyl)aminomethane, and 2.5mL of hydrochloric acid to double-distilled water to a final volume of 100mL to obtain a mixed solution with pH 8.0, which is then stored at 4°C for later use.

[0053] The preparation method of the second labeling reagent includes: 164 μg N-(3-azidopropyl)biotinamine, added to DMSO to 50 μL to obtain a DMSO solution of N-(3-azidopropyl)biotinamine; 1248 μg copper sulfate pentahydrate, added to water to 100 μL to obtain an aqueous solution of copper sulfate pentahydrate; 1430 μg tris(2-carboxyethyl)phosphine hydrochloride, added to water to 100 μL to obtain an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride; 265.3 μg tris[(1-benzyl-1H-1,2,3-triazol-4-yl] [Methyl]amine was added to DMSO to 250 μL to obtain a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; a second labeling reagent was prepared by mixing a DMSO solution of N-(3-azidopropyl)biotinamine, an aqueous solution of copper sulfate pentahydrate, an aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride, and a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, and stored at 4 °C for later use.

[0054] The blocking reagent is 0.1M N-ethylmaleimide.

[0055] The concentration of horseradish peroxidase antibody in the fifth solution is 1 mg / mL.

[0056] The farnesyl inhibitor is 10 mM N-[4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl]-(S)-methionine methyl ester hydrochloride.

[0057] The method for detecting S-geranyl-modified proteins includes the following steps: Add the sixth solution (working concentration of 10 µM) to the cell culture medium and culture for 1 h, then add the first solution and culture for 12 h. Wash the cells and centrifuge to obtain cell pellet. Add 190 μL of the second solution and 10 μL of 0.1 M benzyl sulfonyl fluoride (PMSF) to the obtained cell pellet to lyse the cells. Sonicate at 300 W for 5 seconds, pause for 5 seconds, continue for 2 min, centrifuge at 12000 g, 4 °C for 5 min, collect the supernatant, measure the concentration using the BCA method, take 500 μg of the supernatant into a 0.5 mL EP tube, and dilute with PBS to 180 μL to obtain the protein solution. Add 20 μL of the third solution to the obtained protein solution, invert at room temperature for 1.5 h, then add 15 μL of the fourth solution, invert at room temperature for 4 h to obtain the mixture; Add 600 μL of pre-cooled methanol, 150 μL of chloroform, and 400 μL of ddH2O to the obtained mixture, centrifuge at 4℃ and 6000 rpm for 15 min. A clearly visible milky white membrane is visible in the middle of the solution. Aspirate the upper and lower layers of liquid from the milky white membrane, add 600 μL of pre-cooled methanol, centrifuge at 4℃ and 6000 rpm for 5 min to remove the upper methanol layer, allow the precipitate to air dry naturally for 2-3 min, add 200 μL of the second solution, shake to mix and dissolve the precipitate, and obtain the protein solution. Measure the protein concentration to verify the Western blot (WB).

[0058] The method for verifying WB includes the following steps: The protein solution was separated by SDS-PAGE gel separation and the protein was transferred onto a PVDF membrane; Wash the protein-containing PVDF membrane twice, 10 minutes each time; Prepare a 3% BSA solution with 1XTBS-T and seal the cleaned PVDF membrane at room temperature for 1 hour. Clean the sealed PVDF membrane with 1XTBS-T for 10 min; Prepare a 3% BSA solution with a working concentration using 1XTBS-T, add the fifth solution with a dilution ratio of 1:10000, and incubate the membrane overnight at 4°C. The PVDF membrane was cleaned three times with 1XTBS-T, each time for 10 minutes. Detection was performed using a chemiluminescence analyzer.

[0059] Example 3 A detection kit for S-isoprenylated modified proteins, the detection kit comprising a first solution, a second solution, a third solution, a fourth solution, a fifth solution, and a seventh solution, wherein the first solution is a first labeling reagent, a MAlk-FOH probe; the second solution is a lysis buffer; the third solution is a second labeling reagent; the fourth solution is a blocking reagent; the fifth solution is a horseradish peroxidase antibody; and the seventh solution is a geranyl inhibitor.

[0060] The S-isoprenylated modified protein is an S-farnesyl modified protein.

[0061] The MAlk-FOH probe is prepared by selectively oxidizing the terminal allyl group of farnesol to obtain a functionalized farnesol derivative; then, allyl alcohol is alkylated with propyne bromide and deprotected in acidic ethanol to generate Alk-FOH. The MAlk-FOH probe is stored at 4°C after preparation.

[0062] The working concentration of the MAlk-FOH probe is 50 µM.

[0063] The preparation method of the lysis buffer includes adding 48.048g of urea, 302.85g of tris(hydroxymethyl)aminomethane, and 2.5mL of hydrochloric acid to double-distilled water to a final volume of 100mL to obtain a mixed solution with pH 8.0, which is then stored at 4°C for later use.

[0064] The preparation method of the second labeling reagent includes: 164 μg N-(3-azidopropyl)biotinamine, added to DMSO to 50 μL to obtain a DMSO solution of N-(3-azidopropyl)biotinamine; 1248 μg copper sulfate pentahydrate, added to water to 100 μL to obtain an aqueous solution of copper sulfate pentahydrate; 1430 μg tris(2-carboxyethyl)phosphine hydrochloride, added to water to 100 μL to obtain an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride; 265.3 μg tris[(1-benzyl-1H-1,2,3-triazol-4-yl] [Methyl]amine was added to DMSO to 250 μL to obtain a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; a second labeling reagent was prepared by mixing a DMSO solution of N-(3-azidopropyl)biotinamine, an aqueous solution of copper sulfate pentahydrate, an aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride, and a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, and stored at 4 °C for later use.

[0065] The blocking reagent is 0.1M N-ethylmaleimide.

[0066] The concentration of horseradish peroxidase antibody in the fifth solution is 1 mg / mL.

[0067] The gerany inhibitor is 10 mM (S)2-(4-(((R)-2-amino-3-mercaptopropyl)amino)-2-(naphth-1-yl)benzamido)-4-methylvalerate trifluoroacetate.

[0068] The method for detecting S-farnesyl modified proteins includes the following steps: Add the seventh solution (working concentration of 5 µM) to the cell culture medium and culture for 1-2 h, then add the first solution and culture for 12 h. Wash the cells and centrifuge to obtain the cell pellet. Add 190 μL of the second solution and 10 μL of 0.1 M benzyl sulfonyl fluoride (PMSF) to the obtained cell pellet to lyse the cells. Sonicate at 300 W for 5 seconds, pause for 5 seconds, continue for 2 min, centrifuge at 12000 g, 4 °C for 5 min, collect the supernatant, measure the concentration using the BCA method, take 500 μg of the supernatant into a 0.5 mL EP tube, and dilute with PBS to 180 μL to obtain the protein solution. Add 20 μL of the third solution to the obtained protein solution, invert at room temperature for 1.5 h, then add 15 μL of the fourth solution, invert at room temperature for 4 h to obtain the mixture; Add 600 μL of pre-cooled methanol, 150 μL of chloroform, and 400 μL of ddH2O to the obtained mixture, centrifuge at 4℃ and 6000 rpm for 15 min. A clearly visible milky white membrane is visible in the middle of the solution. Aspirate the upper and lower layers of liquid from the milky white membrane, add 600 μL of pre-cooled methanol, centrifuge at 4℃ and 6000 rpm for 5 min to remove the upper methanol layer, allow the precipitate to air dry naturally for 2-3 min, add 200 μL of the second solution, shake to mix and dissolve the precipitate, and obtain the protein solution. Measure the protein concentration to verify the Western blot (WB).

[0069] The method for verifying WB includes the following steps: The protein solution was separated by SDS-PAGE gel separation and the protein was transferred onto a PVDF membrane; Wash the protein-containing PVDF membrane twice, 10 minutes each time; Prepare a 3% BSA solution with 1XTBS-T and seal the cleaned PVDF membrane at room temperature for 1 hour. Clean the sealed PVDF membrane with 1XTBS-T for 10 min; Prepare a 3% BSA solution with a working concentration using 1XTBS-T, add the fifth solution with a dilution ratio of 1:10000, and incubate the membrane overnight at 4°C. The PVDF membrane was cleaned three times with 1XTBS-T, each time for 10 minutes. Detection was performed using a chemiluminescence analyzer.

[0070] Performance testing I. The detection results of the detection kits in Examples 1 and 2 are shown below. Figure 1 and Figure 2 .

[0071] Depend on Figure 1 It can be seen that the kit for detection using the MAlk-FOH probe proposed in this application is qualified and effective.

[0072] Depend on Figure 2It is known that the detection kit with added geranyl inhibitor has a different banding pattern than the kit without inhibitor, which can detect farnesyl-modified proteins and distinguish between farnesyl-modified and geranyl-modified proteins.

[0073] II. 1. Enrichment of the proteins detected in Example 1 1.1 Add 20 μL of magnetic beads to the obtained protein solution. After equilibrating the magnetic beads with lysis buffer, add 1 mg of peptide sample and shake at low speed at 4 °C overnight for enrichment.

[0074] 1.2. After enrichment, wash 5 times with 500 μL PBS.

[0075] 1.3. After washing, add 50-70 μL of elution buffer (5 mM TCEP in 25 mM ammonium bicarbonate), incubate at 37°C for 15 min. Centrifuge at 15000 g for 1 min at room temperature and collect the supernatant.

[0076] 2. Proteolytic enzyme digestion Add trypsin to the supernatant at a mass ratio of 1:50, and incubate overnight at 37°C and 1000 rpm using a constant temperature mixer. After overnight incubation, centrifuge at room temperature, 12000g, for 8-10 minutes to obtain the peptide.

[0077] 3. Desalting and freeze-drying The sample peptides were purified by desalting with a C18 tip, pre-equilibrated with 50 vol% ACN, washed with 0.1 vol% trifluoroacetic acid, and the C18 tip was slowly and repeatedly pipetted into the sample dissolved in 0.1 vol% trifluoroacetic acid to ensure that the C18 was fully bound to the sample. After binding, the peptides were washed with 0.1% trifluoroacetic acid / 5% ACN and finally eluted with 100 μL of a solution containing 50% acetonitrile and 0.1% trifluoroacetic acid.

[0078] The desalted and purified polypeptide sample was concentrated and evaporated under vacuum.

[0079] 4. Liquid chromatography-tandem mass spectrometry Inverting column information: C18 column (1.8m, 0.15×1.00mm) Chromatography instrument: Thermo Fisher Easy-nLC 1000 Mass spectrometer: Thermo Fisher LTQ Obitrap ETD Chromatographic separation time: 98 min Mobile phase A: 0.1 vol% aqueous formic acid Mobile phase B: 100 vol% acetonitrile solution Flow rate: 600 nL / min The gradient elution procedure is shown in Table 1 below: Table 1

[0080] The mass spectrometry identification parameters are shown in Table 2 below: Table 2

[0081] 5. Data Analysis Raw mass spectrometry data The raw mass spectrometry data were in RAW format and were searched and analyzed using Proteome Discoverer 1.4 (Thermo) software.

[0082] database Data used in this study: FASTA format files from the Human Protein Database. Search settings The database search was performed using Thermo Fisher Scientific's Maxquant 2.1 software, with the relevant parameters shown in Table 3 below: Table 3

[0083] 6. Experimental Results The classic isopentenyl-modified proteins identified are shown in Table 4 below, and their mass spectra are shown in [reference needed]. Figure 4-6 This proves that the test kit in this application is qualified and effective.

[0084] Table 4

Claims

1. A test kit comprising, The detection kit comprises a first solution, a second solution, a third solution, a fourth solution, a fifth solution, the first solution is a first labeling reagent MAIk-FOH probe, the second solution is a lysis solution, the third solution is a second labeling reagent, the fourth solution is a blocking reagent, and the fifth solution is a horseradish peroxidase antibody.

2. The test kit according to claim 1, characterized in that, The detection kit further comprises a sixth solution, and the sixth solution is a farnesyl inhibitor.

3. The test kit according to claim 1, characterized in that, The detection kit further comprises a seventh solution, and the seventh solution is a geranyl inhibitor.

4. The test kit according to claim 1, characterized in that, The pH value of the lysis solution is 7.5-8.5; the lysis solution comprises urea, tris-hydroxymethyl aminomethane, hydrochloric acid and double-distilled water.

5. The test kit according to claim 1, characterized in that, The second labeling reagent comprises a DMSO solution of N-(3-azidopropyl) biotin amine, a copper sulfate pentahydrate aqueous solution, a tris(2-carboxyethyl) phosphine hydrochloride aqueous solution and a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine.

6. Use of a test kit according to any one of claims 1 to 5, characterized in that, It is applied to detect S-isoprenyl modified proteins.

7. Use of a test kit according to claim 2, characterized in that, It is applied to detect S-geranyl modified proteins.

8. Use of a test kit according to claim 3, characterized in that, It is applied to detect S-farnesyl modified proteins.

9. A method of using the test kit according to any one of claims 1-5, characterized in that, The method comprises the following steps: The first solution is added to the cell culture medium, the cells are cultured, the cells are washed, and the cell precipitate is obtained by centrifugation; The second solution and benzylsulfonyl fluoride are added to the obtained cell precipitate to lyse the cells, ultrasonic crushing is performed, the supernatant is obtained by centrifugation, the concentration is measured, and the protein solution is obtained by dilution; The third solution is added to the obtained protein solution, and the solution is inverted at room temperature for 1-3 hours; then the fourth solution is added, and the solution is inverted at room temperature for 1-5 hours to obtain a mixed solution; Precooled methanol, chloroform and ddH2O are added to the obtained mixed solution, and after centrifugation, a clear and visible milky white film is visible in the middle of the solution; the upper and lower liquids of the milky white film are sucked out, precooled methanol is added, centrifugation is performed, the upper methanol is removed, the precipitate is air-dried, the second solution is added to the precipitate to shake and mix, the protein concentration is measured, and WB is verified.

10. The method of using the test kit of claim 9, wherein, The method for verifying the WB comprises the following steps: The protein solution is subjected to SDS-PAGE gel separation, and the protein is transferred to a PVDF membrane; The PVDF membrane with the protein is washed; A BSA solution is prepared, and the above washed PVDF membrane is blocked at room temperature; The blocked PVDF membrane is washed; A BSA solution is prepared, the fifth solution is added, and the membrane is incubated at 4°C overnight; The incubated PVDF membrane is washed; Chemiluminescence exposure detection is performed.

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