A method for in situ labeling and analysis of photoactivatable reagents and proteins

Abstract

The application discloses a photo-crosslinking probe and an in-situ labeling method of G-quadruplex-related proteins based on photo-crosslinking, and an analysis method thereof, the probe can target G-quadruplex, and is covalently crosslinked with G-quadruplex-related proteins in-situ under excitation of ultraviolet light; subsequently, the probe capturing the proteins is biotinylated through a click reaction, and G-quadruplex-related proteins are enriched by using streptavidin agarose microspheres. The application has the advantages that experimental operation is simple and fast, high-specificity in-situ enrichment of G-quadruplex-related proteins can be realized, and important technical support is provided for G-quadruplex in-situ proteomics research.

Description

Technical Field

[0001] A photocrosslinking probe and a photocrosslinking-based in-situ labeling and analysis method for G-quadruplex-related proteins are disclosed. The probe targets G-quadruplexes and undergoes in-situ covalent crosslinking with G-quadruplex-related proteins under ultraviolet light excitation. Subsequently, the probe, containing the captured proteins, is biotinylated via a click reaction, and the G-quadruplex-related proteins are enriched using streptavidin agarose microspheres. The advantages of this invention are its simple and rapid experimental operation, enabling highly specific in-situ enrichment of G-quadruplex-related proteins, and providing important technical support for in-situ proteomics research on G-quadruplexes. Background Technology

[0002] G-quadruplexes are specialized secondary structures of DNA or RNA. In biology, G-quadruplexes have been extensively studied and are believed to play important physiological functions in cellular processes. G-quadruplex-related proteins are a class of proteins involved in regulating the formation, unwinding, or stabilization of G-quadruplexes, playing crucial physiological roles in maintaining genome stability and gene regulation. The functions of these proteins in cells are closely related to the formation and unwinding of DNA secondary structures. Current research shows that G-quadruplexes not only participate in the regulation of gene transcription but also play important roles in post-transcriptional mRNA processing and translation. Furthermore, G-quadruplexes and their related proteins play significant roles in the occurrence and development of hereditary diseases and cancer. Abnormalities in the G-quadruplex structure of specific genes may lead to abnormal gene expression or impaired gene stability, thus being associated with the pathogenesis of hereditary diseases. In addition, abnormalities in the stability and expression levels of G-quadruplexes are also observed in some cancer cells, making G-quadruplexes potential targets for anticancer drugs. Therefore, research on G-quadruplexes and their related proteins is of great significance for advancing basic science, improving disease treatment, and applying them to biotechnology and biomedical research. With a deeper understanding of its mechanism of action, we can expect to see more significant breakthroughs in this field in the future, leading to new applications and developments in science and medicine.

[0003] The emergence of protein chemical labeling technology has provided a powerful tool for achieving highly specific and high-resolution spatial proteomics analysis. Photocrosslinking, as a rapid, simple, and spatiotemporally controllable crosslinking tool, is widely used in various research fields such as chemistry, biology, medicine, and materials science. In chemical biology analysis, photocrosslinking is a powerful tool for studying protein-protein interactions (PPIs) and protein labeling. This patent utilizes photocrosslinking-based G-quadruplex-targeted protein probes at the cellular level, employing 365nm ultraviolet light excitation to induce in-situ covalent crosslinking between the protein probe and G-quadruplex-related proteins. Then, the probe, capturing the protein, is biotinylated via a click reaction and enriched using streptavidin agarose microspheres. This invention enables highly specific in-situ enrichment of G-quadruplex-related proteins, which can be used to study the molecular mechanisms of G-quadruplexes in biosynthesis and cell death, elucidate the molecular mechanisms of G-quadruplex-related diseases, and develop drugs targeting G-quadruplexes and their related proteins, providing crucial technical support for in-situ G-quadruplex proteomics research. Summary of the Invention

[0004] This invention relates to a photocrosslinking probe and a method for enriching G-quadruplex-related proteins based on photocrosslinking. The key feature is that the probe can target G-quadruplexes and undergo in-situ covalent crosslinking with G-quadruplex-related proteins under ultraviolet light excitation. Subsequently, the probe, containing the captured proteins, is biotinylated via a click reaction, and the G-quadruplex-related proteins are enriched using streptavidin agarose microspheres. The advantages of this invention are its simple and rapid experimental operation, enabling highly specific in-situ enrichment of G-quadruplex-related proteins, providing important technical support for proteomics research on G-quadruplex-related proteins.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] (1) Design and synthesize a photocrosslinking probe targeting G-tetrachain. The probe consists of the following three components: 1) a photocrosslinking group—bisacrylidine; 2) a G-tetrachain targeting small molecule ligand; and 3) an enrichment group—alkynyl group.

[0007] (2) The probe undergoes in-situ covalent cross-linking with G-quadruplex-related proteins under ultraviolet light excitation. The process is as follows:

[0008] A. Prepare a 2-10 mM photocrosslinking probe solution using PBS buffer. After incubating the cells in the photocrosslinking probe solution for 2-20 min, the probe will accumulate in the G-quadruplexes of the cells. Then, place the cells on ice and irradiate them with UV light for 2-20 min, followed by incubation in an incubator for 30-60 min. After centrifuging the cell solution, remove the supernatant and wash 1-6 times with pre-cooled PBS buffer to obtain a cell pellet. Add a final concentration of 1-4% SDS solution to the cell pellet, repeatedly pipette and sonicate using a cell sonicator to obtain a cell lysate.

[0009] B. Prepare a photocrosslinking probe solution with a final concentration of 2-10 mM using PBS buffer. Place tissue sections in a culture dish, add the photocrosslinking probe solution, incubate the cells for 2-20 min, then irradiate with ultraviolet light for 2-20 min, and incubate the tissue sections in an incubator for 30-60 min. Add a final concentration of 1-4% SDS solution to the tissue sections, homogenize the tissue thoroughly using a tissue homogenizer, and sonicate using a cell sonicator to obtain cell lysis buffer.

[0010] C. Prepare a probe solution with a final concentration of 2-10 mM using physiological saline solution, and inject the protein probe solution into the animal via injection. 5-10 minutes after injection, irradiate the mouse with ultraviolet light for 2-20 minutes. After 30-60 minutes of irradiation, remove the animal tissue. Add a 1-4% SDS solution to the tissue sample, homogenize the tissue thoroughly using a tissue homogenizer, and then sonicate it using a cell sonicator to obtain cell lysate.

[0011] (3) The probes capturing proteins were biotinylated via a click reaction, and then enriched using streptavidin agarose microspheres to obtain G-quadruplex-related proteins. The procedure was as follows: 1-5 mM azobiotin-azide solution, 5-10 mM sodium ascorbate solution, 20-50 mM THPTA solution, and 50-100 mM copper sulfate solution were added to the cell lysis buffer and reacted at 25-60°C for 1-6 h. Then, an equal volume of 50% streptavidin agarose microsphere solution was added and reacted at 1-4°C for 10-16 h or at 20-25°C for 4-6 h. The mixture was then washed 4-6 times with PBS buffer to remove non-specific adsorption.

[0012] (4) The obtained G-quadruplex-related protein samples were digested on-beads to obtain peptide samples, which were then identified by mass spectrometry and processed. The process was as follows: trypsin enzyme solution was prepared using ammonium bicarbonate buffer, and trypsin enzyme solution was added to the microsphere solution at a mass ratio of 1:25 to 1:50 between trypsin enzyme and G-quadruplex-related protein. The solution was then placed in a constant temperature shaker at 36.5-37.5℃ and an oscillation frequency of 500-1200 rpm for 12-16 h to obtain peptide samples. The peptide samples were separated and analyzed using nano LC-MS, and crystal form analysis and identification were performed using the pFind 2.0 search engine. The advantages of this invention are that the experimental operation is simple and fast, and it can achieve highly specific in-situ enrichment of G-quadruplex-related proteins, providing important technical support for in-situ proteomics research of G-quadruplexes.

[0013] The present invention has the following advantages:

[0014] 1. The experimental procedure is simple, time-saving, and high-throughput, enabling large-scale identification of G-quadruplex-related proteins at the cellular level.

[0015] 2. Under optimal physiological conditions, highly specific and in situ enrichment of G-quadruplex-related proteins can be achieved.

[0016] 3. The photocrosslinking probe used in this invention can label G-quadruplex-related proteins with low energy and only 5 minutes of irradiation time. Therefore, this invention causes minimal disturbance to biological samples.

[0017] 4. This invention is applicable to the enrichment of various G-quadruplex-related proteins from cultured cell lines, tissue cells, tissue sections, and live animals.

[0018] 5. This invention can be used to study the molecular mechanisms of G-quadruplex-related processes in energy metabolism, biosynthesis and cell death, elucidate the molecular mechanisms of G-quadruplex-related diseases, and develop G-quadruplex-related target drugs. Detailed Implementation Plan

[0019] Example 1: Enrichment experiment of G-quadruplex-associated proteins in cultured HeLa cells

[0020] Formula II is the molecular structure of the photocrosslinking probe.

[0021] In the formula, n takes the form of a positive integer 1.

[0022] The following formula represents the synthesis route for photocrosslinking probes:

[0023]

[0024] The specific synthesis steps are as follows:

[0025] 1) Dissolve 3-(2-carboxyethyl)-2-methyliodobenzothiazolium (500 mg, 1.65 mmol), 3-(3-butyl-1-yl)-3H-diazine-3-ethylamine (272.22 mg, 1.99 mmol), 1-hydroxybenzotriazole (224 mg, 1.65 mmol), diisopropylethylamine (214 mg, 1.65 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (318 mg, 1.65 mmol) in 20 mL of DMF (N,N-dimethylformamide), stir overnight at room temperature for 12 h, and monitor the reaction by HPLC (method: the mobile phase acetonitrile:water (containing 0.1% TFA) solution was increased linearly from a volume ratio of 5:95 to 50:50 for 0-50 min, and the product retention time was 23-24 min). Separation and purification were performed using a semi-preparative liquid chromatography system (0-50 min, mobile phase acetonitrile:water (containing 0.1% TFA by volume) solution, with the volume ratio gradually increased from 20:80 to 60:40). The eluent was collected after 22-23 min and freeze-dried under vacuum for 24 h to obtain red solid 1.

[0026] 2) N-((1Z,3E)-2-bromo-3-(phenylimino)prop-1-en-1-yl)aniline (380 mg, 1 mmol), benzothiophene-2-boronic acid (196 mg, 1.1 mmol), Pd(dppf)Cl2 (35 mg, 0.05 mmol), and K2CO3 (415 mg, 3 mmol) were dissolved in 12 mL of 1,4-dioxane / H2O (v / v 5 / 1) and stirred at 85 °C for 1 hour under N2 atmosphere. Finally, the solvent was evaporated and the residue was purified by column chromatography (silica gel packing; eluent: n-hexane / AcOEt = 5 / 1, v / v) to give yellow solid 2.

[0027] 3) Compound 2 (50 mg, 0.14 mmol), Compound 1 (139 mg, 0.29 mmol), and anhydrous NaOAc (46 mg, 0.56 mmol) were dissolved in methanol (2 mL) and heated and stirred at 60 °C for 2 hours under N2 atmosphere. The progress of the reaction was monitored by TLC (volume ratio, MeOH / CH2Cl2 1 / 20, Rf = 0.25). After the reaction was complete, the mixture was cooled, and the mixture was added dropwise to 20 mL of 5% (w / v) KI aqueous solution and stirred for 10 minutes. Finally, the mixture was filtered to obtain the crude product. The crude product was collected by filtration and purified by column chromatography (silica gel; eluent: MeOH / CH2Cl2 = 1 / 100, volume ratio) to obtain the final product shown in Formula II. The structure of the product was characterized by NMR and mass spectrometry and was consistent with the above structural formula.

[0028] (1) Design and synthesis of a photocrosslinking probe targeting G-quadruplexes. The probe comprises three components: a photocrosslinking group—bisacrylidine; a G-quadruplex targeting ligand; and an enrichment group—an alkynyl group. The specific structure is shown in the formula, and a compound synthesized as shown in Formula II is used as the photocrosslinking probe.

[0029] (2) HeLa cell pretreatment. A 5mM photocrosslinking probe solution was prepared using 10mM PBS buffer (pH=7.4). The culture medium in a 6cm culture dish containing 1e7 HeLa cells was removed, and 5mL of the 5mM photocrosslinking probe solution was added. The cells were incubated for 5min, then placed on ice and treated with an energy density of 25μW / cm². 2 The cells were irradiated with ultraviolet light at a wavelength of 365 nm for 10 min. Subsequently, the cells were incubated in an incubator containing 5% CO2 at 37°C for 60 min.

[0030] (3) Cell lysis. Centrifuge the cell solution at 1000×g for 5 min. Remove the supernatant and add 5 mL of pre-chilled 10 mM PBS buffer (pH=7.4) at 0-4℃. Use a pipette to disperse the cell pellet and centrifuge at 1000×g for 5 min. Repeat the process (add 5 mL of pre-chilled 10 mM PBS buffer (pH=7.4) at 0-4℃, disperse the cell pellet, and centrifuge) 3 times to obtain a washed cell pellet. Add 1 mL of 4% wt SDS (sodium dodecyl sulfate) solution to the cell pellet, repeatedly pipette, and sonicate for 2 min using a cell sonicator to obtain a cell lysate.

[0031] (4) Protein enrichment. Four times the volume of pre-cooled -20°C acetone solution was added to the cell lysate to precipitate the protein. The protein precipitate was redissolved using 8M urea aqueous solution to a final protein concentration of 2 mg / mL. To the protein redissolved solution, a final concentration of 5 mM azobiotin-azide (20 mM in H2O, Sigma-Aldrich 900891), a final concentration of 10 mM sodium ascorbate (50 mM), a final concentration of 50 mM tris(3-hydroxypropyltriazine)amine (THPTA, 160 mM), and a final concentration of 100 mM copper sulfate (1 M) solution were added. The mixture was reacted at 60°C for 6 h to obtain the treated solution. Subsequently, an equal volume of 50% streptavidin agarose microsphere solution (Thermo Scientific) was added. TM Pierce TMAvidin Agarose 20219 was reacted at 4°C for 16 h (or at 25°C for 6 h). Afterwards, the mixture was washed with 1 mL of 10 mM PBS buffer (pH 7.4) to remove non-specific adsorption.

[0032] (5) Protein Analysis and Identification. A 20% trypsin enzyme solution was prepared using 50 mM ammonium bicarbonate buffer (pH = 7.4). Trypsin was added to the microspheres at a mass ratio of 1:50 to trypsin and G-quadruplex-related proteins (proteins adsorbed on the microspheres). The solution was then placed in a constant-temperature shaker at 37°C and 1200 rpm for 12 hours to obtain peptide samples. The peptide samples were analyzed using LC-MS. The mass spectrometry results were analyzed and identified using the pFind 2.0 search engine. The found proteins were annotated on the Uniprot website. The proportion of G-quadruplex-related proteins among the total identified proteins was calculated, reaching approximately 30%. Additionally, the found G-quadruplex-related proteins were annotated on the Uniprot website. The proportion of identified G4-related proteins among the total reported G-quadruplex-related proteins was calculated. Nineteen G4-related proteins were identified, accounting for approximately 25% of the total reported G4-related proteins.

[0033] Example 2: Quantitative analysis of G-quadruplex-related proteins in mouse liver cancer tissue sections

[0034] The probes used and their synthesis were the same as in Example 1.

[0035] (1) Design and synthesis of photocrosslinking probe targeting G-tetrachain. The probe consists of the following three components: a photocrosslinking group—bisacrylidine; a G-tetrachain targeting ligand; and an enrichment group—an alkynyl group. The specific structure is shown in Formula II, and the compound synthesized in Formula II is used as the photocrosslinking probe.

[0036] (2) Tissue section pretreatment. A 5mM photocrosslinking probe solution was prepared using 10mM PBS buffer (pH=7.4). Control group (normal human liver tissue sections) and human hepatocellular carcinoma tissue sections were placed in culture dishes, and 10mL of the 5mM photocrosslinking probe solution was added. After incubation for 5 min, the cells were placed on ice and incubated with an energy density of 25μW / cm². 2 The cells were irradiated with ultraviolet light at a wavelength of 365 nm for 10 min. Subsequently, the cells were incubated in a 5% CO2 incubator at 37°C for 60 min.

[0037] (3) Cell lysis. The tissue was centrifuged to remove the supernatant, and 3 mL of 4% wtSDS solution was added to the sample. The mouse tissue was thoroughly broken up using a tissue homogenizer and then sonicated for 2 min using a cell sonicator to obtain cell lysate.

[0038] (4) Protein enrichment. Four times the volume of pre-cooled -20°C acetone solution was added to the cell lysis buffer to precipitate the protein. The protein precipitate was redissolved using 8M urea to a final concentration of 2 mg / mL. A final concentration of 5 mM azobiotin-azide (20 mM), a final concentration of 10 mM sodium ascorbate (50 mM), a final concentration of 50 mM THPTA (160 mM), and a final concentration of 100 mM copper sulfate (1 M) solution were added to the cell lysis buffer, and the reaction was carried out at 60°C for 6 h. Subsequently, an equal volume of 50% streptavidin agarose microspheres (Thermo Scientific) were added. TM Pierce TM Avidin Agarose 20219 was reacted at 4°C for 16 h or at 25°C for 6 h. Afterwards, the mixture was washed with 2 mL of 10 mM PBS buffer (pH 7.4) to remove non-specific adsorption.

[0039] (5) Protein Analysis and Identification. Trypsin enzyme solution (20% by mass) was prepared using ammonium bicarbonate buffer. Trypsin enzyme was added to microspheres at a mass ratio of 1:50 to G-quadruplex-related proteins. The solution was then placed in a constant-temperature shaker at 37°C and 1200 rpm for 12 hours to obtain peptide samples. Peptide samples were analyzed using LC-MS. The pFind 2.0 search engine was used to analyze and identify the mass spectrometry results. The found proteins were annotated on the Uniprot website. The proportion of G-quadruplex-related proteins among the total identified proteins was calculated, reaching approximately 25%. Additionally, the found G-quadruplex-related proteins were annotated on the Uniprot website. The proportion of identified G4-related proteins among the total reported G-quadruplex-related proteins was calculated. Seventeen G4-related proteins were identified, accounting for approximately 20% of the total reported G4-related proteins.

Claims

1. A photoactivating agent, characterized in that, Use one or more of the photoactivating reagents having the general structural formula shown in Formula I: Formula I In the formula, n takes one or more positive integers, such as 1, 2, or 3.

2. A method for in situ labeling and analysis of proteins, characterized in that: Using the photoactivating reagent described in claim 1 as a probe, in situ labeling and / or analysis of G-quadruplex interaction proteins based on photocrosslinking is performed. The photoactivating reagent includes the following three characteristic components: (1) a photocrosslinking group—bisacrididine; (2) a G-quadruplex targeting ligand; and (3) an enrichment group—alkynyl group. It is a photocrosslinking probe targeting parallel G-quadruplexes, with its photocrosslinking group being diacaridine. This group, upon photoactivation by ultraviolet light, generates a highly reactive carbene intermediate that can covalently bind to proteins under physiological conditions. This group operates at energy densities of 25-10000 μW / cm². 2 Full activation can be achieved under ultraviolet light irradiation; the G-quadruplex targeting ligand is a cyanine dye ligand with high selectivity for parallel G-quadruplexes as reported; the enrichable group of the probe is alkynyl, which is suitable for any click chemistry-based enrichment strategy, including the introduction of biotin through click chemistry reaction, followed by enrichment reaction using streptavidin agarose microspheres; the in-situ labeling and analysis method is for non-disease treatment and diagnosis.

3. The method according to claim 2, characterized in that, This method is applicable to the in situ labeling and enrichment of G-quadruplex-binding proteins in biological systems with normal functional activity. The biological system is selected from one or more of the following: cultured cell lines, tissue cells, tissue sections, or living animals.

4. The method according to claim 2 or 3, characterized in that, This method, through in-situ labeling and enrichment of G-quadruplex-binding proteins, can be used to study the molecular mechanisms of G-quadruplex in biosynthesis and / or cell death, elucidate the molecular mechanisms of G-quadruplex-related diseases, or develop drugs targeting G-quadruplex and its related proteins.

5. The method according to any one of claims 4, characterized in that, The specific process is as follows: 1) The probe is covalently cross-linked with G-quadruplex-related proteins in situ under ultraviolet light excitation, and the cells are then lysed to obtain cell lysate; 2) The probes that capture proteins are biotinylated by click reaction, and then enriched using streptavidin agarose microspheres to obtain G-quadruplex-related proteins. 3) The obtained G-quadruplex-related protein samples were digested with on-beads to obtain peptide samples, which were then identified by mass spectrometry and processed.

6. The method according to claim 5, characterized in that, The process by which the probe undergoes in-situ covalent cross-linking with G-quadruplex-related proteins under ultraviolet light excitation is as follows: A. Prepare a 2-10 mM photocrosslinking probe solution using PBS buffer; After incubating cells in photocrosslinked probe solution for 2-20 min, the probe will accumulate in the G-quadruplexes of the cells. Subsequently, the cells are placed on ice and irradiated with ultraviolet light for 2-20 min, followed by incubation in an incubator for 30-60 min. After centrifugation of the cell solution, the supernatant is removed and the cells are washed 1-6 times with pre-cooled PBS buffer at 0-4℃ to obtain a cell pellet. After adding 1-4% SDS solution to the cell pellet, the cell pellet is pipetted and then sonicated using a cell sonicator to obtain a cell lysate. Alternatively, B. Prepare a photocrosslinking probe solution with a final concentration of 2-10 mM using PBS buffer; place the tissue section in a culture dish, add the photocrosslinking probe solution, incubate the cells for 2-20 min, then irradiate with ultraviolet light for 2-20 min, and incubate the tissue section in an incubator for 30-60 min; add 1-4% SDS solution to the tissue section, use a tissue homogenizer to thoroughly break up the tissue, and use a cell sonicator to sonicate to obtain cell lysate; Alternatively, C. Prepare a probe solution with a final concentration of 2-10 mM using physiological saline solution, and inject the protein probe solution into the animal tissue required for the study by injection; 5-10 minutes after injection, irradiate the corresponding tissue of the animal with ultraviolet light for 2-20 minutes. After 30-60 minutes of light exposure, the animal tissue was removed. A 1-4% SDS solution was added to the tissue sample, and the tissue was thoroughly broken up using a tissue homogenizer. The tissue was then sonicated using a cell sonicator to obtain cell lysate.

7. The method according to claim 6, characterized in that: The probes capturing proteins are biotinylated via a click reaction, and then enriched using streptavidin agarose microspheres to obtain G-quadruplex-related proteins. The process is as follows: 1-5 mM azobiotin-azide solution, 5-10 mM sodium ascorbate solution, 20-50 mM THPTA solution, and 50-100 mM copper sulfate solution are added to the obtained cell lysate, and the mixture is reacted at 25-60°C for 1-6 h; subsequently, an equal volume of 45-55% (v / v) streptavidin agarose microsphere solution is added, and the mixture is reacted at 1-4°C for 10-16 h or at 20-25°C for 4-6 h; finally, the mixture is washed 4-6 times with PBS buffer to remove non-specific adsorption.

8. The in-situ labeling and analysis method according to claim 5, characterized in that: The obtained G-quadruplex-related protein samples were digested on-beads to obtain peptide samples, which were then identified and processed by mass spectrometry. The procedure was as follows: trypsin enzyme solution was prepared using ammonium bicarbonate buffer, and trypsin enzyme solution was added to streptavidin agarose microspheres at a mass ratio of trypsin enzyme to G-quadruplex-related protein of 1:25 to 1:

50. The solution was then placed in a thermostatic shaker at a temperature of 36.5-37.5℃ and a shaking frequency of 500-1200 rpm for 12-16 h to obtain peptide samples. The peptide samples were analyzed using LC-MS.

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