A probe for labeling a protein, and a preparation method and application thereof

Abstract

The application relates to the field of chemical proteomics, in particular to a probe for labeling protein, and a structure formula of the probe is shown as formula I; the probe realizes protein labeling by labeling active amino acids, wherein the active amino acids are tyrosine, lysine, serine and threonine. The probe provides a new auxiliary tool for covalent small molecule drugs, protein covalent ligand screening, drug or active natural product target discovery and elucidation of protein-protein interaction. By using the probe and combining mass spectrometry analysis technology, high-throughput detection is realized, so that the analysis difficulty is reduced, and the experimental period is shortened; in addition, the probe is suitable for complex biological samples, and protein does not need to be purified, and the operation is simple.

Description

Technical Field

[0001] This application relates to the field of chemical proteomics, specifically to a probe for labeling proteins. Background Technology

[0002] Chemical proteomics is a protein annotation technique built on the interdisciplinary fields of synthetic chemistry, proteomics, and cell biology. It uses specialized chemical probes with different functions as core tools, combined with high-resolution mass spectrometry, to directly analyze the activity, drug binding, and binding sites of proteins under physiological conditions in living cells or tissues.

[0003] Chemical probes are small molecule compounds known to modulate specific protein targets in specific ways. These probes are used in drug discovery and development. In a typical drug discovery workflow, after high-throughput or fragment screening is used to identify compounds that affect the target in a certain way (lead compounds), and after promising lead compounds are developed into lead candidates using medicinal chemistry methods, chemical probes are typically used for further screening. By improving our understanding of targets and pathways, chemical probes can have a significant impact on facilitating and accelerating the discovery and development of new drugs, reducing risks before the time and expense of clinical trials. Furthermore, these chemical probes can identify molecular targets or pathway mechanisms, study protein function, protein-protein interactions, and more. Specifically, chemical probes can be used to help establish relationships between molecular targets and the broader biological outcomes of regulating those targets in cells or organisms. They can also help discover new biology associated with the target, elucidate the relationship between the target and phenotype, and validate whether a specific target is an appropriate intervention point affecting disease progression or outcome.

[0004] The design and development of chemical probes based on active molecules or known small molecule ligands has certain limitations. Probes can only target specific proteins, resulting in low target accessibility. Furthermore, probes need to be designed one by one based on active molecules or known small molecule ligands, leading to low throughput.

[0005] Therefore, developing a universal probe based on quantitative chemical proteomics technology, combined with mass spectrometry analysis for high-throughput protein identification and analysis, is of great significance for the discovery of small molecule ligands and even drugs for which there are currently no known druggable targets. Summary of the Invention

[0006] Technical issues

[0007] Traditional methods for designing and developing chemical probes based on active molecules or known small molecule ligands and using chemical proteomics to analyze and identify target proteins and mechanisms of action are limited by the need for individual probe design and synthesis, resulting in probes targeting only specific proteins, low target accessibility, and low throughput. Traditional methods for confirming whether a small molecule interacts with a protein require protein purification; identifying the specific binding site of a small molecule often requires single- or multi-point amino acid mutations, which are inherently inaccurate and time-consuming. Furthermore, many proteins, especially membrane proteins, are difficult to purify. This invention aims to provide a universal probe based on quantitative chemical proteomics technology. This probe can be combined with mass spectrometry for high-throughput protein identification and analysis, and can be widely used in drug screening, identification of drug or active natural product targets, elucidation of mechanisms of action, and protein-protein interactions.

[0008] Technical solution

[0009] The first aspect of this application provides a probe for labeling proteins, the probe having the structural formula shown in Formula I:

[0010]

[0011] In some embodiments, the probe labels the protein by labeling an active amino acid; wherein the active amino acid is tyrosine, lysine, serine, and threonine.

[0012] A second aspect of this application provides a method for preparing a probe for the above-mentioned labeled protein, comprising the following steps:

[0013]

[0014] In the step of preparing compound II from compounds IV and III, the condensing agent is carbodiimide hydrochloride and the catalyst is 1-hydroxybenzotriazole.

[0015] In some embodiments, in the step of preparing compound II from compounds IV and III, the organic base is one of triethylamine or N,N-diisopropylethylamine; in the step of preparing compound I from compound II, the organic acid is formic acid or acetic acid.

[0016] In some embodiments, in the step of preparing compound II from compounds IV and III, the organic base is one of triethylamine or N,N-diisopropylethylamine; the molar ratio of compound III, compound IV and organic base is 1:1.1 to 1.5:3 to 6.

[0017] In some embodiments, in the step of preparing compound I from compound II, the catalyst is boron trifluoride-diethyl ether or aluminum trichloride; the organic acid is formic acid or acetic acid.

[0018] The third aspect of this application provides an application of the probe of the above-mentioned labeled protein in the identification of active molecular target proteins.

[0019] In some embodiments, the method for identifying the target protein of the active molecule is as follows: first, the active molecule is co-incubated with cells for cell drug delivery; then, the probe is co-incubated with cells for protein labeling; then, the cells are collected and lysed; then, the probe-labeled protein is biotinylated using a biotinylated compound; finally, the probe-labeled protein is separated from the proteome using streptavidin-coupled magnetic beads; and the probe-labeled protein is analyzed by mass spectrometry. By identifying the changes in the degree of target protein probe labeling caused by the active molecule, the target protein of the active molecule can be identified in the cell system.

[0020] In some embodiments, the specific steps of cell drug delivery are as follows: washing cells with PBS and serum-free culture medium respectively, and culturing cells in a serum-free culture medium containing 1-500 μM of active molecules of the target to be identified in an incubator for 0.5-1 h.

[0021] In some embodiments, the specific steps of protein labeling are as follows: washing cells with PBS and serum-free culture medium respectively, and culturing cells in a serum-free culture medium containing 100-500 μM probe in an incubator for 0.5-1 h.

[0022] In some embodiments, the specific steps of the biotin modification are as follows: adding biotin azide compound with a final concentration of 100-200M, sodium ascorbate with a final concentration of 1-2.5mM, BTTAA with a final concentration of 10-25mM, and copper sulfate (CuSO4) with a final concentration of 6-12.5mM to the probe-labeled cell lysate in sequence, and reacting the mixed solution for 1-1.5 hours.

[0023] In some embodiments, the specific steps for separating the probe-labeled protein from the proteome are as follows: 200–300 μL of streptavidin-conjugated magnetic beads are added to the protein precipitation buffer and incubated by rotation at room temperature for 2–3 h. After enrichment, the magnetic beads are resuspended in 6–9 M Urea / 100 mM triethanolamine borate, reacted with 10–15 mM dithiothreitol for 15–30 min, 25–40 mM indole-3-acetic acid for 15–30 min, and 15–25 mM dithiothreitol for 15–30 min, followed by trypsin digestion overnight.

[0024] The fourth aspect of this application provides an application of the probe of the above-mentioned labeled protein in drug screening.

[0025] In some embodiments, the drug screening method specifically involves: utilizing the principle that the target protein probes of the added compound group and the blank group have different labeling degrees, using the probes of the labeled proteins as described in claim 1 to perform protein labeling, and combining mass spectrometry-based quantitative proteomics technology to identify the changes in the target protein probe labeling degree caused by the compounds, thereby achieving the screening of the compound library in the cell system.

[0026] The fifth aspect of this application provides a kit containing a probe comprising the aforementioned labeled protein.

[0027] Technical effect

[0028] This invention discloses a universal probe capable of labeling active tyrosine, lysine, serine, and threonine residues in proteins, providing a novel auxiliary tool for screening covalent small molecule drugs and protein covalent ligands, discovering drug or active natural product targets, and elucidating protein-protein interactions. Specifically, using the probe of this invention in conjunction with mass spectrometry enables high-throughput detection, thereby reducing analytical difficulty and shortening the experimental cycle. Furthermore, the probe of this invention is suitable for complex biological samples, requires no protein purification, and is simple to operate. Attached Figure Description

[0029] Figure 1 For probe I 1 HNMR spectrum;

[0030] Figure 2 The F NMR spectrum of probe I;

[0031] Figure 3 The MS spectrum of probe I is shown below.

[0032] Figure 4 This is a schematic diagram illustrating the principle of probe I labeling active amino acids;

[0033] Figure 5 The image shows the fluorescence signal of the gel image of the protein labeled with probe I;

[0034] Figure 6 The image shows the Coomassie blue staining signal of the gel image of the protein labeled with probe I;

[0035] Figure 7 This is a schematic diagram of the experimental procedure for protein identification using probe I;

[0036] Figure 8 A scatter plot showing the screening results of 100 covalent compounds. Detailed Implementation

[0037] To facilitate the explanation of the technical solution of this application, the following is a general explanation and definition of the terms and expressions used in this application.

[0038] The terms “comprising,” “including,” or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase “comprising one…” does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0039] In the comparative experiments provided in this application, unless otherwise specified, all other experimental conditions and materials are kept consistent to ensure comparability.

[0040] Unless otherwise specified, all reagents and instruments used in the embodiments of this invention can be purchased from the market.

[0041] The following provides a further description of a protein-labeling probe, its preparation method, and its application.

[0042] Example 1: Synthesis of Probe I

[0043]

[0044] 4-Amino-1-naphthic acid (580 mg, 3.1 mmol) was dissolved in DMF (10 mL), and compound N was added sequentially with stirring. 1 -Methyl-N 1 -Propylethynylethylenediamine (347 mg, 2.34 mmol), triethylamine (TEA, 940 mg, 9.3 mmol), 1-hydroxybenzotriazole (HOBT, 837 mg, 6.2 mmol), and carbodiimide hydrochloride (EDCI, 1.19 g, 6.2 mmol). The mixture was stirred at 25 °C for 12 hours, and LC-MS showed complete consumption of 4-amino-1-naphthic acid. The reaction mixture was concentrated under reduced pressure, and the residue was subjected to reversed-phase preparative chromatography (column: Biotech XPt C). 18 The sample was separated and purified to obtain a yellow oily iodonitrile tetrazolium chloride (INT, 315 mg), namely compound II, using a mobile phase of [water (10 mM NH4HCO3)-acetonitrile] and a gradient of 10%-40% acetonitrile.

[0045] To a solution of INT (150 mg, 533 μmol) in acetonitrile (2 mL), boron trifluoride-diethyl ether (BF3·OEt2, 98.4 μL, 799 μmol) and tert-butyl nitrite (t-BuONO, 76.1 μL, 639 μmol) were added, and the mixture was stirred at 25 °C for 1 hour. Under N2 protection, potassium metabisulfite (K2S2O5, 296 mg, 1.33 mmol), N-fluorobis(benzenesulfonamide) (NFSI, 252 mg, 799 μmol), water (1 mL), and acetic acid (AcOH, 2 mL) were added sequentially. The mixture was heated to 50 °C and stirred for 11 hours. LC-MS showed complete consumption of INT. The reaction mixture was concentrated under reduced pressure, and the residue was subjected to preparative thin-layer chromatography (ethyl acetate:MeOH = 10:1, R0). f =0.7) separation and purification yielded the crude product. The crude product was then subjected to reversed-phase preparative chromatography (column: Waters Xbridge BEH C). 18 The probe was separated and purified using a 100*30mm, 10μm filter; mobile phase: [water (10mM NH4HCO3)-acetonitrile]; gradient: 35%-65% acetonitrile) to obtain a yellow oily probe I (1.7mg).

[0046] Example 2: Synthesis of Probe I

[0047] 4-Amino-1-naphthic acid (655 mg, 3.5 mmol) was dissolved in DMF (10 mL), and compound N was added sequentially with stirring. 1 -Methyl-N 1 -Propylethynylethylenediamine (347 mg, 2.34 mmol), N,N-diisopropylethylamine (DIPEA, 1806 mg, 14 mmol), 1-hydroxybenzotriazole (HOBT, 837 mg, 6.2 mmol), and carbodiimide hydrochloride (EDCI, 1.19 g, 6.2 mmol). The mixture was stirred at 25 °C for 12 hours, and LC-MS showed complete consumption of 4-amino-1-naphthic acid. The reaction mixture was concentrated under reduced pressure, and the residue was subjected to reversed-phase preparative chromatography (column: Biotech XPt C). 18 The sample was separated and purified to obtain a yellow oily iodonitrile tetrazolium chloride (INT, 315 mg) and compound II. The mobile phase was [water (10 mM NH4HCO3)-acetonitrile]; gradient: 10%-40% acetonitrile.

[0048] To a solution of INT (150 mg, 533 μmol) in acetonitrile (2 mL), boron trifluoride-diethyl ether (BF3·OEt2, 98.4 μL, 799 μmol) and tert-butyl nitrite (t-BuONO, 76.1 μL, 639 μmol) were added, and the mixture was stirred at 25 °C for 1 hour. Under N2 protection, potassium metabisulfite (K2S2O5, 296 mg, 1.33 mmol), N-fluorobisbenzenesulfonamide (NFSI, 252 mg, 799 μmol), water (1 mL), and formic acid (2 mL) were added sequentially. The mixture was heated to 50 °C and stirred for 11 hours. LC-MS showed complete consumption of INT. The reaction mixture was concentrated under reduced pressure, and the residue was subjected to preparative thin-layer chromatography (ethyl acetate:MeOH = 10:1, R0). f =0.7) separation and purification yielded the crude product. The crude product was then subjected to reversed-phase preparative chromatography (column: Waters Xbridge BEH C). 18 The probe was separated and purified using a 100*30mm, 10μm filter; mobile phase: [water (10mM NH4HCO3)-acetonitrile]; gradient: 35%-65% acetonitrile) to obtain a yellow oily probe I (1.7mg).

[0049] Example 3: Characterization of Probe I

[0050] Probe I was characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The proton NMR spectrum is shown below. Figure 1 As shown, the fluorine spectrum is as follows Figure 2 As shown, the mass spectrometry is as follows Figure 3 As shown. The results are as follows:

[0051] HNMR: 1 H NMR (400MHz, DMSO-d6)δ=8.82-8.71(m,1H),8.53-8.49(m,1H),8.47-

[0052] 8.41(m,1H),8.35(d,J=8.4Hz,1H),7.96(t,J=7.6Hz,1H),7.89-7.82(m,1H),7.77(d,J=7.6Hz,1H),3.49-3.43(m,2H),3.40(br d,J=2.0Hz,2H),3.16(br d,J=2.4Hz,1H),2.65-2.57(m,2H),2.35-2.27(m,3H).

[0053] FNMR: 19 F NMR (376MHz, DMSO-d6) δ = -73.41 (s, 1F).

[0054] MS(ESI+): m / z 349.1 [M+H] +.

[0055] Example 4: Labeling of live cell proteins by probe I

[0056] 1) Preparation of cell lines, culture media and test compounds

[0057] Table 1. Composition of culture media used for each cell line

[0058]

[0059] As shown in the table, the cell lines to be tested were placed in a culture medium containing 10% heat-inactivated FBS (fetal bovine serum), 2 mM L-glutamine, 100 U / mL penicillin, and 100 g / mL streptomycin, and cultured in a cell culture incubator at 37°C and 5% CO2. The medium was changed every two days. After the cells reached 80% confluence, they were trypsinized, passaged, and kept in a good logarithmic growth phase.

[0060] 2) Detection method

[0061] Cells in the logarithmic growth phase grow at a rate of 5*10 6 Cells were seeded per 10cm cell culture dish and cultured for 24 hours. Cells were washed with PBS and serum-free medium, respectively. Serum-free medium containing probes (100μM and 500μM) was prepared and added to the culture dishes. Cells were then incubated for 0.5–1 hour. The cell culture dishes were then placed on ice and washed twice with pre-chilled PBS (4°C). Cells were then scraped off and collected using PBS.

[0062] The obtained cells were added to cell lysis buffer (PBS, 1% IGEPA-CA-630, 0.2% SDS, 1% EDTA-free protease inhibitor mixture, 0.1% Benzonase), sonicated, and then centrifuged at high speed (20000g, 30min) in a benchtop centrifuge at 4℃. The supernatant was collected and the concentration was adjusted to 2mg / mL with cell lysis buffer. 50μL of protein solution was added to a final concentration of 200M fluorescein azide (Rhodamine-azide), a final concentration of 2.5mM sodium ascorbate (Sigma-Aldrich), a final concentration of 25mM BTTAA (Xi'an Kangfuno Biotechnology Co., Ltd.), and a final concentration of 12.5mM CuSO4 (Tianjin Runjing Energy Technology Co., Ltd.) at room temperature, and reacted for 1h. After the reaction, the samples were separated by SDS-PAGE and electrophoresed at 150V for approximately 1h. The separated proteins were visualized by scanning the gel with a ChemiDoc imaging system (model: Bio-Rad Chemidoc MP) to display fluorescent bands, followed by Coomassie blue staining. A schematic diagram of probe I labeling with active amino acids is shown below. Figure 4 As shown. The results of this embodiment are as follows. Figure 5 (fluorescence signal results) and Figure 6 (Results of Coomassie blue staining are shown).

[0063] Example 5: Omics analysis of probe I labeled proteins

[0064] To identify the types of proteins and amino acid residues labeled by the probe, the applicant conducted chemical proteomics analysis at the probe binding site level and the protein level. (See flowchart.) Figure 7 As shown, after adding probe labels to cells, the cells were lysed. A "click chemistry" reaction was used to couple a biotinylate-cleavable tag to the probe-labeled protein, enriching the probe-labeled protein. The protein peptide sample (probe-labeled protein sample) was obtained by enzymatic digestion and separation of the supernatant. Then, the beads were acid-hydrolyzed and the supernatant was separated to obtain probe-modified peptide samples (probe-labeled peptide samples). The peptide samples were then subjected to high-resolution mass spectrometry analysis, and the data were searched in a library for quantitative analysis, as detailed below:

[0065] 1) Preparation of cell lines, culture media and test compounds

[0066] For cell line types, culture medium components used for each cell line, and cell culture methods, please refer to step 1 of Example 4.

[0067] 2) Detection method

[0068] Cells in the logarithmic growth phase grow at a rate of 5*10 6 Cells were seeded per 10cm cell culture dish and cultured for 24 hours. Cells were washed with PBS and serum-free medium, respectively. Serum-free medium containing 500μM probe was prepared and added to the culture dishes. Cells were then incubated for 0.5–1 hour. The cell culture dishes were then placed on ice and washed twice with pre-cooled PBS (4°C). Cells were then scraped off and collected using PBS.

[0069] The obtained cells were added to cell lysis buffer (PBS, 1% IGEPA-CA-630, 0.2% SDS, 1% EDTA-free protease inhibitor mixture, 0.1% Benzonase), sonicated, and then centrifuged at high speed (20000g, 30min) in a benchtop centrifuge at 4℃. The supernatant was collected and the concentration was adjusted to 2mg / mL with cell lysis buffer. 500μL of protein solution was added to a final concentration of 200M biotin-Dadps-azide, a final concentration of 2.5mM sodium ascorbate, a final concentration of 25mM BTTAA, and a final concentration of 12.5mM CuSO4 at room temperature, and reacted for 1h. After the above reaction was completed, the protein solution was transferred to a 15mL centrifuge tube, and 4 volumes of methanol, vortexed and mixed thoroughly, 1 volume of chloroform, vortexed and mixed thoroughly, and 3 volumes of water, vortexed and mixed thoroughly. Centrifuge at 3000 rpm for 10 min at 4°C, retaining the protein precipitate. Add 1 mL of pre-cooled (4°C) methanol, vortex or sonicate (for about 5 seconds) to break up the protein precipitate, then transfer to a 1.5 mL centrifuge tube, centrifuge at 3000 rpm for 3 min at 4°C, and discard the supernatant. Then, add another 1 mL of pre-cooled (4°C) methanol, vortex or sonicate (for about 5 seconds) to break up the protein precipitate, then transfer to a 1.5 mL centrifuge tube, centrifuge at 3000 rpm for 3 min at 4°C, and discard the supernatant. Afterward, air-dry the protein precipitate for 2 min to completely remove the methanol.

[0070] The protein precipitate was resuspended in 3 mL of 0.05% SDS / PBS, and 200 μL of streptavidin-conjugated magnetic beads were added and incubated at room temperature for 3 h. After enrichment, the magnetic beads were resuspended in 6 M Urea / 100 mM triethanolamine borate (TEAB), reacted with 10 mM dithiothreitol (DTT) for 30 min, 25 mM indole-3-acetic acid (IAA) for 30 min, and 15 mM MTT for 30 min, followed by trypsin digestion overnight. The supernatant was collected after digestion, labeled with tandem mass spectrometry (TMT), and the samples were combined, fractionated, and then evaporated to dryness to obtain probe-labeled protein samples. The magnetic beads were resuspended in 2% (v / v) formic acid-water solution, incubated at room temperature for 20 min, the supernatant was collected, desalted, and then evaporated to dryness to obtain probe-labeled peptide samples.

[0071] The probe-labeled protein samples and probe-labeled peptide samples were resuspended in 20 μL of 0.1% (v / v) formic acid aqueous solution and analyzed using LC-MS / MS. The obtained mass spectrometry data were then searched using protein sequence libraries in conventional proteomics analysis software (such as Maxquant, ProteinDiscovery, Spectronaut, etc.) (e.g., downloaded from Uniprot). Appropriate screening criteria were set in the software used (screening criteria for samples with probe-labeled sites: peptide presence with quantitative value, PSM>0, Qvality PEP≤0.01, and Modifications including probe modification; screening criteria for samples without probe-labeled peptides: peptide presence with quantitative value, #Protein Unique Peptides≥2, theory coverage≥0.5, and Contaminant=FALSE). Reliable protein results for probe labeling were obtained through screening, and the sequence information of the obtained labeled peptides was analyzed. The detection results of this embodiment are shown in Tables 2 and 3. This invention tested peptides containing probe-labeled amino acids and peptides of probe-labeled protein samples after probe incubation of cells. The results showed that probe I can label and quantify more than 2,600 peptides of 135 proteins, including tyrosine, lysine, tryptophan and threonine.

[0072] Table 2. Labeled peptides in HEK293T cells

[0073]

[0074] The sequence information of the obtained labeled peptides was analyzed, and the proteins to which the labeled peptides belonged were determined based on their abundance. The information of the 135 identified proteins is shown in Table 3.

[0075] Table 3. Labeled proteins in HEK293T cells

[0076]

[0077]

[0078] Example 6: Identification of active molecular targets using probe I

[0079] Identification principle: Based on the principle that the target protein probes in the active molecule group and the blank group are labeled with different degrees, the probes of this invention are used to label proteins and combined with mass spectrometry-based quantitative proteomics technology to identify the changes in the target protein probe labeling degree caused by the active molecules, so as to realize the identification of the target proteins of active molecules in the cell system.

[0080] The remaining experimental procedures are the same as in Example 5, except that:

[0081] Cell drug administration before probe labeling: Wash cells with PBS and serum-free medium, respectively. Prepare serum-free medium containing DMSO and serum-free medium containing 1-500 μM of the active molecule of the target to be identified, respectively, add them to cell culture dishes, and incubate the cells in an incubator for 0.5-1 h.

[0082] Identification and analysis process: The peptide sample is subjected to high-resolution mass spectrometry analysis, the data is searched in a database, quantitative analysis is performed, and the occupancy rate is calculated using the following formula:

[0083]

[0084] Among them, Intensity C Indicates the mass spectrometry signal of the probe-labeled peptide in the blank (DMSO) group; Intensity T This represents the mass spectrometry signal of the probe-labeled peptide of the active molecular group of the target to be identified.

[0085] Example 7: The rest is the same as Example 5, except that:

[0086] Probe labeling steps: Probe labeling is performed using serum-free culture medium containing 350 μM probe.

[0087] The steps for biotin modification are as follows: Take 50 μL of protein solution and add 100 M fluorescein azide compound, 1 mM sodium ascorbate, 10 mM BTTAA, and 6 mM CuSO4 at room temperature. React for 1.5 h.

[0088] The steps for separating probe-labeled proteins from the proteome are as follows: 300 μL of streptavidin-conjugated magnetic beads are added to the protein precipitation buffer and incubated by rotation at room temperature for 2 h. After enrichment, the magnetic beads are resuspended in 9 M Urea / 100 mM triethanolamine borate, reacted with 15 mM dithiothreitol for 15 min, 40 mM indole-3-acetic acid for 15 min, and 25 mM dithiothreitol for 15 min, and then digested with trypsin overnight.

[0089] Example 8: Drug screening using probe I

[0090] Drug screening principle: Based on the principle that the target protein probes of the compound group and the blank group are labeled with different degrees, the probes of this invention are used to label proteins and combined with mass spectrometry-based quantitative proteomics technology to identify the changes in the target protein probe labeling degree caused by the compound, thereby realizing the screening of compound libraries in the cell system.

[0091] Everything else is the same as in Example 5, except that:

[0092] Cell drug administration before probe labeling: Cells were washed with PBS and serum-free medium, respectively. Serum-free medium containing DMSO and serum-free medium containing 1-500 μM of the compound to be screened were prepared and added to two sets of cell culture dishes. The cells were then incubated with the drug in an incubator for 1 h.

[0093] The group with serum-free medium containing DMSO was the blank group, and the group with serum-free medium containing 1-500 μM of the compound to be screened was the compound group.

[0094] Identification and analysis process: The peptide sample is subjected to high-resolution mass spectrometry analysis, the data is searched in a database, quantitative analysis is performed, and the occupancy rate is calculated using the following formula:

[0095]

[0096] Among them, Intensity C Indicates the mass spectrometry signal of the probe-labeled peptide in the blank (DMSO) group; Intensity T This represents the mass spectrometry signal of the probe-labeled peptide in the group of compounds (molecules in the compound library) to be screened.

[0097] A screening was conducted on a library of covalent compounds containing 100 molecules. The scatter plot of the screening results is shown below. Figure 8 .

[0098] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.

Claims

1. A probe for labeling proteins, characterized in that, The probe structure is shown in Formula I:

2. The probe for labeling proteins according to claim 1, characterized in that, The probe labels proteins by labeling them with active amino acids; wherein the active amino acids are tyrosine, lysine, serine, and threonine.

3. A method for preparing a probe for a labeled protein as described in claim 1, characterized in that, Includes the following steps: ； In the step of preparing compound II from compounds IV and III, the condensing agent is carbodiimide hydrochloride, and the catalyst is 1-hydroxybenzotriazole.

4. The preparation method according to claim 3, characterized in that, In the step of preparing compound II from compounds IV and III, the organic base is either triethylamine or N,N-diisopropylethylamine; the molar ratio of compound III, compound IV and organic base is 1:1.1 to 1.5:3 to 6.

5. The preparation method according to claim 3, characterized in that, In the step of preparing compound I from compound II, the catalyst is boron trifluoride-diethyl ether or aluminum trichloride; the organic acid is formic acid or acetic acid.

6. The use of the probe for the labeled protein as described in claim 1 in the identification of active molecular target proteins.

7. The application according to claim 6, characterized in that, The method for identifying the target protein of the active molecule is as follows: First, the active molecule is co-incubated with cells for cell drug delivery. Then, the probe is co-incubated with cells for protein labeling. Next, the cells are collected and lysed. Then, the probe-labeled protein is biotinylated using a biotinylated compound. Finally, the probe-labeled protein is separated from the proteome using streptavidin-coupled magnetic beads. The probe-labeled protein is then analyzed by mass spectrometry. By recognizing the changes in the degree of target protein probe labeling caused by the active molecule, the target protein of the active molecule can be identified in the cell system.

8. The application according to claim 7, characterized in that, The specific steps for cell drug delivery are as follows: wash cells with PBS and serum-free culture medium respectively, and culture cells in an incubator for 0.5 to 1 hour using serum-free culture medium containing 1 to 500 μM of active molecules of the target to be identified.

9. The application according to claim 7, characterized in that, The specific steps for protein labeling are as follows: wash cells with PBS and serum-free culture medium respectively, and culture cells in an incubator for 0.5 to 1 hour using serum-free culture medium containing 100 to 500 μM probe.

10. The application according to claim 7, characterized in that, The specific steps of the biotin modification are as follows: add biotin azide compound with a final concentration of 100-200M, sodium ascorbate with a final concentration of 1-2.5mM, BTTAA with a final concentration of 10-25mM, and copper sulfate with a final concentration of 6-12.5mM to the probe-labeled cell lysate in sequence, and react the mixed solution for 1-1.5 hours.

11. The application according to claim 7, characterized in that, The specific steps for separating the probe-labeled protein from the proteome are as follows: 200-300 μL of streptavidin-conjugated magnetic beads are added to the protein precipitation buffer and incubated by rotation at room temperature for 2-3 h. After enrichment, the magnetic beads are resuspended in 6-9 M Urea / 100 mM triethanolamine borate, reacted with 10-15 mM dithiothreitol for 15-30 min, 25-40 mM indole-3-acetic acid for 15-30 min, and 15-25 mM dithiothreitol for 15-30 min, followed by trypsin digestion overnight.

12. The application of the probe for the labeled protein as described in claim 1 in drug screening.

13. The application according to claim 12, characterized in that, The specific method for drug screening is as follows: utilizing the principle that the target protein probes of the added compound group and the blank group have different labeling degrees, the probes of the labeled proteins described in claim 1 are used for protein labeling, and combined with mass spectrometry-based quantitative proteomics technology to identify the changes in the target protein probe labeling degree caused by the compound, thereby realizing the screening of the compound library in the cell system.

14. A kit comprising a probe of the labeled protein as claimed in claim 1.

Images