Photoactivation reagent and in-situ labeling and analysis method of protein

Abstract

The invention discloses a photo-crosslinking probe, a G-quadruplex related protein in-situ marker based on photo-crosslinking and an analysis method of the G-quadruplex related protein in-situ marker based on photo-crosslinking. The probe can target a G-quadruplex and is subjected to in-situ covalent crosslinking with the G-quadruplex related protein under the excitation of ultraviolet light; and then, biotinylating the probe which captures the protein through a click reaction, and enriching the G-quadruplex related protein by using streptavidin agarose microspheres. The method has the advantages that the experimental operation is simple and quick, the high-specificity in-situ enrichment of the G-quadruplex related protein can be realized, and an important technical support is provided for the in-situ proteomics research of the G-quadruplex.

Description

Technical Field

[0001] A photo-crosslinking probe and an in situ labeling of G-quadruplex-related proteins based on photo-crosslinking and an analysis method thereof, characterized in that: the probe can target G-quadruplexes and covalently crosslink with G-quadruplex-related proteins in situ under ultraviolet light excitation; subsequently, the probe that captures the protein is biotinylated by a click reaction, and G-quadruplex-related proteins are enriched using streptavidin agarose microspheres. The invention has the advantages of simple and quick experimental operation, can achieve highly specific in situ enrichment of G-quadruplex-related proteins, and provides important technical support for in situ proteomics research of G-quadruplexes. Background Art

[0002] G-quadruplex is a special secondary structure of DNA or RNA. In biology, G-quadruplex has been widely studied and is believed to play an important physiological function in cellular processes. G-quadruplex-related proteins are a class of proteins involved in regulating the formation, unwinding or stabilization of G-quadruplexes. They play a key physiological function 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 studies have shown that G-quadruplexes are not only involved in the regulation of gene transcription, but also play an important role in post-transcriptional mRNA processing and translation. At the same time, G-quadruplexes and their related proteins play an important role in the occurrence and development of genetic diseases and cancer. Abnormal G-quadruplex structure of specific genes may lead to abnormal expression or impaired stability of genes, which is related to the pathogenesis of genetic diseases. In addition, the stability and expression level of G-quadruplexes in some cancer cells also show abnormalities, which makes G-quadruplexes a potential target for anticancer drugs. Therefore, the study of G-quadruplexes and their related proteins is of great significance for promoting the development of basic science, improving disease treatment, and applying them to biotechnology and biomedical research. As we gain a deeper understanding of its mechanism of action, we can expect to see more important breakthroughs in this field in the future, thereby bringing new applications and developments to science and medicine.

[0003] The emergence of protein chemical labeling technology provides a powerful tool for achieving high-specificity and high-resolution spatial proteomics analysis. Photocrosslinking reaction is widely used in different research fields such as chemistry, biology, medicine and materials as a fast, simple and spatiotemporally controllable crosslinking tool. In chemical biological analysis, photocrosslinking is a powerful tool for studying protein-protein interactions (PPIs), protein labeling and other fields. This patent uses a G-quadruplex-targeted protein probe based on photocrosslinking at the cellular level, and uses ultraviolet light excitation with a wavelength of 365nm to cause the protein probe to covalently crosslink with the G-quadruplex-related protein in situ. Then, the probe that captures the protein is biotinylated by a click reaction and enriched using streptavidin agarose microspheres. The present invention can achieve high-specific in situ enrichment of G-quadruplex-related proteins, which can be used for the molecular mechanism study of G-quadruplex in the process of biosynthesis and cell death, the molecular mechanism interpretation of G-quadruplex-related diseases, and the development of drugs targeting G-quadruplexes and their related proteins, providing important technical support for the in situ proteomics study of G-quadruplexes. Summary of the invention

[0004] The present invention relates to a photo-crosslinking probe and a G-quadruplex-related protein enrichment method based on photo-crosslinking, characterized in that: the probe can target G-quadruplexes and covalently crosslink with G-quadruplex-related proteins in situ under ultraviolet light excitation; subsequently, the probe that captures the protein is biotinylated by a click reaction, and streptavidin agarose microspheres are used to enrich G-quadruplex-related proteins. The present invention has the advantages of simple and fast experimental operation, can achieve high-specificity in situ enrichment of G-quadruplex-related proteins, and provides important technical support for the proteomics research of G-quadruplex-related proteins.

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

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

[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 photo-crosslinking probe solution with a molar concentration of 2-10 mM using PBS buffer; after the cells are placed in the photo-crosslinking probe solution and incubated for 2-20 minutes, the probe will be enriched in the G-quadruplex of the cells; then, place the cells on ice and irradiate with ultraviolet light for 2-20 minutes, and then, place the cells in an incubator and incubate for 30-60 minutes; after centrifuging the cell solution, remove the supernatant solution and add pre-cooled PBS buffer to wash 1-6 times to obtain a cell pellet; add a final concentration of 1-4% SDS solution to the cell pellet and repeatedly blow and sonicate it using a cell ultrasonic disruptor to obtain a cell lysate;

[0009] B. Use PBS buffer to prepare a photo-crosslinking probe solution with a final concentration of 2-10mM. Place the tissue slice in a culture dish, add the photo-crosslinking probe solution, incubate the cells for 2-20 minutes, and irradiate with ultraviolet light for 2-20 minutes. Place the tissue slice in an incubator and incubate for 30-60 minutes. Add a final concentration of 1-4% SDS solution to the tissue slice, use a tissue homogenizer to fully break up the tissue, and use a cell ultrasonic disruptor to obtain a cell lysate.

[0010] C. Prepare a probe solution with a final concentration of 2-10mM using physiological saline solution, and inject the protein probe solution into the animal body. 5-10min after injection, irradiate the mouse with ultraviolet light for 2-20min. After 30-60min of irradiation, remove the animal tissue. Add a final concentration of 1-4% SDS solution to the tissue sample, use a tissue homogenizer to fully break up the tissue, and use a cell ultrasonic disruptor to obtain a cell lysate.

[0011] (3) The probe that captured the protein was biotinylated by click reaction, and then enriched with streptavidin agarose microspheres to obtain G-quadruplex-related proteins. The process is as follows: add azobiotin-azide solution with a final concentration of 1-5mM, sodium ascorbate solution with a final concentration of 5-10mM, THPTA solution with a final concentration of 20-50mM, and copper sulfate solution with a final concentration of 50-100mM to the cell lysate, and react for 1-6h at 25-60℃. Then, add a 50% streptavidin agarose microsphere solution with an equal volume of cell lysate, and react for 10-16h at 1-4℃ or 4-6h at 20-25℃. Then, wash with PBS buffer 4-6 times to remove non-specific adsorption.

[0012] (4) The obtained G-quadruplex-related protein sample is subjected to on-beads enzymatic hydrolysis to obtain a peptide sample, which is then subjected to mass spectrometry identification and data processing. The process is as follows: a trypsin enzyme solution is prepared using an ammonium bicarbonate buffer, and the trypsin enzyme solution is added to the microsphere solution at a mass ratio of trypsin enzyme to G-quadruplex-related protein of 1:25 to 1:50, and the solution is placed in a constant temperature oscillator at a temperature of 36.5-37.5°C and an oscillation frequency of 500-1200rpm, and the peptide sample is obtained by enzymatic hydrolysis for 12-16 hours. The peptide sample is separated and analyzed using nano LC-MS, and the crystal form is analyzed and identified using the pFind 2.0 search engine. The advantages of the present invention are that the experimental operation is simple and fast, and a highly specific in situ enrichment of G-quadruplex-related proteins can be achieved, providing important technical support for in situ proteomics research on G-quadruplexes.

[0013] The present invention has the following advantages:

[0014] 1. The experimental operation steps are simple, time-saving, and high-throughput, which can achieve large-scale identification of G-quadruplex-related proteins at the cellular level.

[0015] 2. While maintaining the optimal physiological state of cells, highly specific and in situ enrichment of G-quadruplex-related proteins can be achieved.

[0016] 3. The photo-crosslinking probe used in the present invention can achieve labeling of G-quadruplex-related proteins with low energy and only 5 minutes of irradiation time. Therefore, the present invention has little disturbance to biological samples.

[0017] 4. The present invention is applicable to the enrichment of various G-quadruplex-related proteins in cultured cell lines, tissue cells, tissue sections, living animals, etc.

[0018] 5. The present invention can be used to study the molecular mechanisms of G-quadruplexes in energy metabolism, biosynthesis and cell death, to explain the molecular mechanisms of G-quadruplex-related diseases, and to develop G-quadruplex-related target drugs. Specific implementation plan

[0019] Example 1: G-quadruplex-related protein enrichment experiment on cultured Hela cells

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

[0021] In the formula, n is a positive integer 1

[0022] The following is the synthesis route of the photocrosslinking probe: The specific synthesis steps are as follows: 1) 3-(2-carboxyethyl)-2-methyliodobenzothiazolium (500 mg, 1.65 mmol), 3-(3-butan-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), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (318 mg, 1.65 mmol) were dissolved in 20 ml DMF (N,N dimethylformamide), stirred at room temperature overnight for 12 h, and the reaction was monitored by HPLC (method: 0-50 min mobile phase acetonitrile: water (containing 0.1% TFA by volume) solution linearly increased from 5:95 volume ratio to 50:50, product retention time 23-24 min). Semi-preparative liquid chromatography was used for separation and purification (the mobile phase was acetonitrile: water (containing 0.1% TFA by volume) solution with a gradient increase from a volume ratio of 20:80 to 60:40 during 0-50 min), and the effluent was collected at 22-23 min and freeze-dried in vacuo for 24 h to obtain a red solid 1. 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)Cl 2 (35 mg, 0.05 mmol) and K 2 CO 3 (415 mg, 3 mmol) was dissolved in 12 mL 1,4-dioxane / H2O (v / v 5 / 1) and heated under N 2 The mixture was stirred at 85° C. for 1 hour under atmosphere. Finally, the solvent was evaporated and the residue was purified by column chromatography (silica gel packing; eluent: n-hexane / AcOEt=5 / 1, volume ratio) to obtain 2 as a yellow solid. 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 stirred at N 2 The mixture was heated and stirred at 60°C for 2 hours under a 3-H atmosphere. The progress of the reaction was determined by TLC (volume ratio, MeOH / CH 2 Cl 2 1 / 20, Rf = 0.25) monitoring. After the reaction was completed, the mixture was cooled and added dropwise to 20 mL of 5% KI aqueous solution and stirred for 10 minutes, and finally filtered to obtain a crude product. The crude product was collected by filtration and purified by column chromatography (silica gel; eluent: MeOH / CH 2 Cl 2=1 / 100, volume ratio) to obtain the final product shown in Formula II. The product structure was consistent with the above structural formula by NMR and mass spectrometry.

[0023] (1) Design and synthesis of a photocrosslinking probe targeting G-quadruplex. The probe includes the following three components: a photocrosslinking group - diazirine; a G-quadruplex targeting ligand; and an enrichable group - alkynyl. The specific structure is shown in the formula, and the compound synthesized as Formula II is used as a photocrosslinking probe.

[0024] (2) Hela cell pretreatment. A photocrosslinking probe solution with a final concentration of 5 mM was prepared using 10 mM PBS buffer (pH = 7.4). The culture medium in the 6 cm culture dish containing 1e7 Hela cells was removed, and 5 mL of 5 mM photocrosslinking probe solution was added. The cells were incubated for 5 min, and then placed on ice and subjected to an energy density of 25 μW / cm 2 The cells were then exposed to ultraviolet light at a wavelength of 365 nm for 10 min. 2 Incubate in an incubator at 37°C with air for 60 min.

[0025] (3) Cell lysis. The cell solution was centrifuged at 1000×g for 5 min. The supernatant solution was removed and 5 mL of 10 mM PBS buffer (pH=7.4) precooled at 0-4°C was added, and the cell precipitate was dispersed using a pipette. The cell solution was centrifuged at 1000×g for 5 min. Repeat (add 5 mL of 10 mM PBS buffer (pH=7.4) precooled at 0-4°C to disperse the cell precipitate, and centrifuge) 3 times to obtain the washed cell precipitate. 1 mL of 4% wt SDS (sodium dodecyl sulfate) solution was added to the cell precipitate, and the solution was repeatedly blown and sonicated for 2 min using a cell ultrasonic disruptor to obtain a cell lysis solution.

[0026] (4) Protein enrichment. Add four times the volume of pre-cooled -20°C acetone solution to the cell lysate to obtain protein precipitation. Use 8M urea aqueous solution to redissolve the protein precipitate to a final protein concentration of 2 mg / mL. Add azobiotin-azide (20 mM in H) to a final concentration of 5 mM to the protein resolubilization solution. 2 O, Sigma-Aldrich 900891) solution, a final concentration of 10mM sodium ascorbate (50mM) solution, a final concentration of 50mM tris (3-hydroxypropyl triazine methyl) amine (THPTA, 160mM) solution and a final concentration of 100mM copper sulfate (1M) solution were reacted at 60°C for 6h to obtain a treated solution. Then, a 50% volume fraction of streptavidin agarose microsphere solution (Thermo Scientific TM PierceTM Avidin Agarose 20219) was added and reacted at 4°C for 16 h (or at 25°C for 6 h). Subsequently, 1 mL of 10 mM PBS buffer (pH = 7.4) was used for washing to remove non-specific adsorption.

[0027] (5) Analysis and identification of proteins. A trypsin enzyme solution (mass concentration of 20%) was prepared using 50 mM ammonium bicarbonate buffer (pH = 7.4), and the trypsin enzyme solution was added to the microspheres at a mass ratio of 1:50 between trypsin enzyme and G-quadruplex-related protein (protein adsorbed on the microspheres). The solution was placed in a constant temperature oscillator at a temperature of 37°C and an oscillation frequency of 1200 rpm for 12 h 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 proteins found were annotated on the Uniprot website, and the proportion of G-quadruplex-related proteins to the total identified proteins was calculated, which was about 30% overall. In addition, the G-quadruplex-related proteins found were annotated on the Uniprot website, and the proportion of the identified G4-related proteins to the total reported G-quadruplex-related proteins was calculated. 19 G4-related proteins were identified, accounting for about 25% of the total number of reported G4-related proteins.

[0028] Example 2: Quantitative analysis of G-quadruplex-related proteins in mouse liver cancer tissue sections

[0029] The probe used and its synthesis are the same as in Example 1.

[0032] ((1) Design and synthesis of a photocrosslinking probe targeting G-quadruplex. The probe includes the following three components: a photocrosslinking group - diazirine; a G-quadruplex targeting ligand; and an enrichable group - alkynyl. The specific structure is shown in Formula II, and the compound synthesized as shown in Formula II is used as a photocrosslinking probe.

[0033] (2) Tissue slice pretreatment. Use 10mM PBS buffer (pH=7.4) to prepare a photo-crosslinking probe solution with a final concentration of 5mM. Place the control group, normal human liver tissue slices and human liver cancer tissue slices, in a culture dish, add 10mL of 5mM photo-crosslinking probe solution, incubate the cells for 5min, place the cells on ice and use an energy density of 25μW / cm 2 The cells were then exposed to ultraviolet light at a wavelength of 365 nm for 10 min. 2 Incubate in an incubator at 37°C with air for 60 min.

[0034] (3) Cell lysis. The tissue was centrifuged to remove the supernatant, 3 mL of 4% wt SDS solution was added to the sample, the mouse tissue was fully broken up using a tissue homogenizer, and the cell was sonicated for 2 min using a cell ultrasonic disruptor to obtain a cell lysate.

[0035] (4) Protein enrichment. Add four times the volume of pre-cooled -20°C acetone solution to the cell lysate to obtain protein precipitation. Use 8M urea to redissolve the protein precipitate, and the final protein concentration is 2 mg / mL. Add azobiotin-azide solution (20mM) with a final concentration of 5mM, sodium ascorbate (50mM) with a final concentration of 10mM, THPTA (160mM) with a final concentration of 50mM, and copper sulfate (1M) with a final concentration of 100mM to the cell lysate, and react at 60°C for 6h. Then, add an equal volume of 50% streptavidin agarose microsphere solution (Thermo Scientific TM Pierce TM Avidin Agarose 20219) was added and reacted at 4°C for 16 h or at 25°C for 6 h. Subsequently, 2 mL of 10 mM PBS buffer (pH = 7.4) was used for washing to remove non-specific adsorption.

[0036] (5) Analysis and identification of proteins. Use ammonium bicarbonate buffer to prepare trypsin enzyme solution (mass concentration of 20%), and add trypsin enzyme solution to the microspheres according to the mass ratio of trypsin enzyme to G-quadruplex-related protein of 1:50. The solution is placed in a constant temperature oscillator at a temperature of 37°C and an oscillation frequency of 1200rpm. The enzymatic hydrolysis is carried out for 12 hours to obtain peptide samples. The peptide samples are analyzed by LC-MS. The mass spectrometry results are analyzed and identified using the pFind 2.0 search engine. The searched proteins are annotated on the Uniprot website, and the proportion of G-quadruplex-related proteins to the total identified proteins is calculated. The overall proportion can reach about 25%. In addition, the searched G-quadruplex-related proteins are annotated on the Uniprot website, and the proportion of the identified G4-related proteins to the total reported G-quadruplex-related proteins is calculated. 17 G4-related proteins were identified, accounting for about 20% of the total number of reported G4-related proteins.

Claims

1. A photoactivated reagent, characterized in that Use one or more of the photoactivated reagents having the general structural formula shown in Formula I: In the formula, n is a positive integer selected from the group consisting of 1, 2, and 3, or two or more thereof.

2. The photoactivatable reagent according to claim 1, characterized in that: The photoactivation reagent comprises the following three characteristic components: (1) a photocrosslinking group, namely, diaziridine; (2) a G-quadruplex targeting ligand; and (3) an enrichable group, namely, an alkynyl group. It is a photo-crosslinking probe targeting parallel G-quadruplexes. Its photo-crosslinking group is diazirine. When the group is irradiated with ultraviolet light, diazirine can be activated by light to produce a highly reactive carbene intermediate, which can covalently bind to proteins under physiological conditions. The group can react with the energy density of 25-10000μW / cm 2 Full activation can be achieved under ultraviolet light irradiation; the G-quadruplex targeting ligand is a cyanine dye ligand that has been reported to be highly selective for parallel G-quadruplexes; the enrichable group of the probe is an alkynyl group, which is suitable for any enrichment strategy based on click chemistry, including the introduction of biotin through click chemistry reaction and then enrichment reaction using streptavidin agarose microspheres.

3. A method for in situ labeling and analysis of proteins, characterized in that: The photoactivatable reagent according to claim 1 or 2 is used as a probe to perform in situ labeling and / or analysis of G-quadruplex-interacting proteins based on photocrosslinking.

4. The method according to claim 3, characterized in that: The method is applicable to in situ labeling and enrichment of G-quadruplex binding proteins in biological systems with normal functional activity (or living), and the biological systems include one or more of cultured cell lines, tissue cells, tissue slices or living animals.

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

6. The method according to any one of claims 3 to 5, characterized in that: The specific process is: 1) The probe is covalently cross-linked with G-quadruplex-related proteins under ultraviolet light excitation, and the cells are broken to obtain cell lysate; 2) The probe that captured the protein was biotinylated by a click reaction, and then enriched using streptavidin agarose microspheres to obtain G-quadruplex-related proteins; 3) The obtained G-quadruplex-related protein samples are subjected to on-beads enzymatic hydrolysis to obtain peptide samples for mass spectrometry identification and data processing.

7. The method according to claim 6, characterized in that The process of in situ covalent cross-linking labeling of the probe with G-quadruplex-related proteins under ultraviolet light excitation is as follows: A. Prepare a photo-crosslinking probe solution with a molar concentration of 2-10 mM using PBS buffer; After the cells are placed in a photo-crosslinked probe solution and incubated for 2-20 minutes, the probe will be enriched in the G-quadruplex of the cells; then, the cells are placed on ice and irradiated with ultraviolet light for 2-20 minutes, and then, the cells are placed in an incubator and incubated for 30-60 minutes; after the cell solution is centrifuged, the supernatant solution is removed and 0-4°C pre-cooled PBS buffer is added to wash 1-6 times to obtain a cell precipitate; after adding a 1-4% SDS solution to the cell precipitate, the cell precipitate is blown with a pipette, and a cell ultrasonic disruptor is used to ultrasonicate to obtain a cell lysate; or B. using PBS buffer to prepare a photo-crosslinking probe solution with a final concentration of 2-10 mM; placing the tissue slice in a culture dish, adding the photo-crosslinking probe solution, incubating the cells therein for 2-20 minutes, and irradiating with ultraviolet light for 2-20 minutes, and incubating the tissue slice in an incubator for 30-60 minutes; adding a 1-4% SDS solution to the tissue slice, using a tissue homogenizer to fully break up the tissue, and using a cell ultrasonic disruptor to ultrasonicate to obtain a cell lysate; or 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; 5-10 minutes after the injection, irradiate the corresponding tissue of the animal with ultraviolet light for 2-20 minutes; After irradiation for 30-60 minutes, the animal tissues are taken out; a 1-4% SDS solution is added to the tissue sample, the tissue is fully broken up using a tissue homogenizer, and a cell ultrasonic disruptor is used to ultrasonicate to obtain a cell lysate.

8. The method according to claim 6 or 7, characterized in that: The probe that captures the protein is biotinylated by a click reaction, and then enriched by streptavidin agarose microspheres to obtain G-quadruplex-related proteins, the process being: adding a 1-5 mM azobiotin-azide solution, a 5-10 mM sodium ascorbate solution, a 20-50 mM THPTA solution, and a 50-100 mM copper sulfate solution to the obtained cell lysate, and reacting at 25-60° C. for 1-6 hours; then reacting with a 45-55% (preferably 50%) volume fraction of streptavidin agarose microsphere solution equal to the volume of the cell lysate at 1-4° C. for 10-16 hours or at 20-25° C. for 4-6 hours; Subsequently, the cells were washed 4–6 times with PBS buffer to remove nonspecific adsorption.

9. The in situ labeling and analysis method according to claim 6, characterized in that: The obtained G-quadruplex-associated protein sample was subjected to on-beads enzymatic hydrolysis to obtain peptide samples, which were then subjected to mass spectrometry identification and data processing. The process 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-associated protein of 1:25 to 1:

50. The solution was placed in a constant temperature oscillator at a temperature of 36.5-37.5°C and an oscillation frequency of 500-1200 rpm. The peptide samples were obtained by enzymatic hydrolysis for 12-16 hours. The peptide samples were analyzed using LC-MS.

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