A Venn-enriched molecular sieve and its application

Abstract

This invention discloses a Venn-enriched molecular sieve and its applications. The Venn-enriched molecular sieve is composed of non-denaturing separating gels with different gradients. The applications include using the Venn-enriched molecular sieve to screen fluorescent small molecule compounds and for small and large molecule compounds that retain biological activity after modification with fluorescent groups. This invention successfully separates, screens, and identifies binding proteins of small molecules (Xc), polysaccharides (Gc), fatty acids (Lc), proteins (Pc), and nucleic acids (Dc) by combining Venn-enriched molecular sieves, subcellular localization of fluorescent compounds, and overlapping protein analysis on gels with different gradients.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a Venn enrichment molecular sieve and its applications. Background Technology

[0002] Small molecule compounds with natural fluorescence have weak protein binding interactions, and the binding between fluorescent small molecule compounds and proteins is disrupted by protein denaturing agents such as SDS and mercaptoethanol. Therefore, common methods such as denatured protein extraction and gel electrophoresis cannot enrich the binding bands of fluorescent small molecule compounds to proteins, making further screening of target proteins impossible. Fluorescent small molecule compounds lose their biological activity after being modified with macromolecular chemical groups such as biotin or fluorescent probes; therefore, methods using chemical modification to screen for target proteins bound to fluorescent small molecule compounds are generally ineffective.

[0003] Compounds that are highly soluble in water retain biological activity even after being modified with fluorescent groups, making it difficult to screen them for target proteins using traditional methods (such as biotin affinity chromatography, photosensitive cross-linking, and chemical cross-linking mass spectrometry).

[0004] Currently, there is no effective experimental method that can both preserve the interaction between fluorescent small molecule compounds and proteins, especially compounds with a molecular weight of less than 1000 Daltons, reduce non-specific interference from non-target proteins through group enrichment, and identify the binding target proteins of fluorescent small molecule compounds without requiring a control group. Therefore, it is crucial to develop a product and method that can solve the above-mentioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a Venn-enriched molecular sieve; the second purpose is to provide applications of the Venn-enriched molecular sieve.

[0006] The first objective of this invention is achieved by the Venn enrichment molecular sieve being composed of non-denaturing separating gels of different gradients.

[0007] This invention employs non-denaturing separating gels of different gradients (8%-15%) as a type of "Venn enrichment molecular sieve." Under a non-denaturing environment, it can enrich the interaction bands between fluorescent small molecule compounds and proteins on separating gels of different gradients. After these bands are cut, protein spectroscopy analysis is performed. Through Venn enrichment, proteins binding to the target fluorescent small molecule compound are screened. The specific Venn enrichment principle and process are as follows: After the fluorescent small molecule compound binds to the target protein, it may further form protein complexes with other proteins in a non-denaturing environment. Therefore, the protein complexes may form protein complexes of different sizes depending on the amount of protein they contain. The non-denaturing gels are divided into 8%, 10%, 12%, and 15% pore sizes according to their pore size from largest to smallest. The protein complexes formed by binding with the fluorescent small molecule compound may be enriched on non-denaturing protein gels of different pore sizes depending on the amount of protein they contain. We first selected proteins that overlapped on the 8%-15% gels for verification. Among them, the more times the protein overlapped on the four gradient gels, the greater the possibility of binding to the target fluorescent compound. If the target fluorescent small molecule compound has a well-defined subcellular localization, such as mitochondria, nucleus, or cell membrane, the screening range of target proteins can be further narrowed. Even if the subcellular localization of the target fluorescent small molecule compound has not yet been discovered, its fluorescence properties can still be used to find it later. In addition, this method of the present invention does not require a control group.

[0008] The second objective of this invention is achieved by using the Venn enrichment molecular sieve for screening fluorescent small molecule compounds and for large molecule compounds such as polysaccharides, fatty acids, proteins, and nucleic acids that still retain biological activity after modification with the CY3 fluorescent group.

[0009] This invention successfully isolated, screened, and identified Xc-binding proteins by combining non-denaturing gradient gels (Venn enrichment molecular sieves), subcellular localization of fluorescent compounds, and analysis of overlapping proteins on different gradient gels. Attached Figure Description

[0010] Figure 1 This is a schematic diagram of the Venn enrichment molecular sieve analysis of the present invention; Figure 2 This is a schematic diagram comparing the Venn enrichment molecular sieve of the present invention with conventional mitochondrial protein enrichment. Figure 3 This is a schematic diagram illustrating the application principle of the Venn enrichment molecular sieve of the present invention. Figure 4 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention; Figure 5 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 2 ; Figure 6This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 3 ; Figure 7 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 4 ; Figure 8 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 5 ; Figure 9 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 6 ; Figure 10 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 7 ; Figure 11 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 8 ; Figure 12 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 9 ; Figure 13 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 10 ; Figure 14 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 10 one; Figure 15 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 10 two; Figure 16 This is a schematic diagram illustrating the application effect of the Venn enrichment molecular sieve of the present invention. Figure 10 three. Detailed Implementation

[0011] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0012] The Venn enrichment molecular sieve described in this invention is composed of non-denaturing separating gels with different gradients.

[0013] The gradient of the non-denaturing separating gel is 8% to 15%.

[0014] The non-deforming adhesives are classified into 8%, 10%, 12%, and 15% according to their pore size from largest to smallest.

[0015] The application of the Venn enrichment molecular sieve described in this invention is to screen fluorescent small molecule compounds and to use small and large molecule compounds that still have biological activity after modification with biotin or photosensitive probes.

[0016] The invention will be further illustrated below with specific implementation examples: Example 1 The fluorescent small molecule compound Xc (Xc is derived from compounds I-IV described in patent CN109528713A) was selected and located in the mitochondria. Xc has a relative molecular mass of 432. Xc has a fluorescent group and emits red fluorescence after being excited by light with a wavelength of 570 nm. The emission wavelength is 610 nm. Xc has anti-tumor activity, but the target is unknown. 1 mM Xc was incubated overnight at 37°C in ovarian cancer cells A2780. Total mitochondrial protein was extracted under non-denaturing conditions and separated by gradient non-denaturing polyacrylamide gel electrophoresis (Native-PAGE). (8%-15% non-denaturing separating gels were prepared in advance, with a 5% non-denaturing stacking gel used as the top layer. No protein denaturing reagents such as SDS or β-mercaptoethanol were added during preparation, protein extraction, and gel running to maintain protein binding with Xc.) Electrophoresis was performed at 150V on ice for 3-4 hours. Electrophoresis was stopped when bromophenol blue just ran off the gel. The gel was then placed in a luminescence analyzer equipped with a fluorescent gel imaging system, and the CY3 channel was used for illumination and imaging. The red bands were excised for mass spectrometry analysis. Based on the mass spectrometry results, Venn diagrams were used to enrich and select target proteins. The target protein was attached to the CM5 chip, and the binding ability of Xc to the target protein was detected and verified using a macromolecular interaction analyzer (BIACORE).

[0017] The formulation of 8%-15% non-denaturing separating gel is shown in the table below: Table 1 Formulation of 8%-15% separating adhesive / block adhesive

[0018] 5% non-denaturing stack gel formulation / block gel formulation: 2.84 mL ultrapure water (ddH2O), 0.83 mL 30% polyacrylamide solution, 1.26 mL 0.5 M Tris solution (pH=6.8), 0.05 mL 10% APS, 0.005 mL TEMED.

[0019] Among them, 1) 30% acrylamide solution formulation: Dissolve 290g acrylamide and 10g N,N-methyleneacrylamide in 600mL of warm deionized water, make up to 1L, filter with a 0.45μm filter membrane and use.

[0020] 2) 1.5M Tris solution (pH=8.8): Dissolve 18.15g Tris in 80mL of ultrapure water, adjust the pH to 8.8 with concentrated hydrochloric acid, and make up the volume to 100mL with ultrapure water.

[0021] 3) 0.5M Tris solution (pH=6.8): Dissolve 12.1g Tris in 80mL of ultrapure water, adjust the pH to 6.8 with concentrated hydrochloric acid, and make up the volume to 100mL with ultrapure water.

[0022] 4) 10% APS formulation: Weigh 0.1g of ammonium persulfate and dissolve it in 1mL of ultrapure water.

[0023] 5) TEMED was purchased from Sigma (item number: 110-18-9).

[0024] 6) Electrophoresis buffer formulation: Weigh 3.02g Tris and 18.8g glycine and dissolve in 1L of ultrapure water.

[0025] Example 2 Application tests of Venn enrichment molecular sieves: I. Test Plan 1. Gradient non-denaturing gel enrichment of target proteins that bind to Xc; 2. Protein spectroscopy analysis; 3. Detection of Xc binding affinity to proteins; 4. Regulation of target protein function by Xc.

[0026] II. Experimental Procedure 1. Prepare 8%, 10%, 12%, and 15% non-denaturing separating gel solutions and 5% non-denaturing stacking gel solutions in 15mL centrifuge tubes. Take 2mL of each concentration solution and add it sequentially to a 1.5mm gap in a glass plate. After the next layer of gel solidifies, add the next layer of gel solution. After adding the gel solutions, press each layer with ethanol. The bottom layer is the 15% non-denaturing separating gel, and the top layer is the 5% non-denaturing stacking gel.

[0027] Dilute Xc to 50 mM with DMSO, add 5 μL of Xc to 1 mL of cell culture medium, and then add 1 × 10⁻⁶ ovarian cancer cells A2780 to the cell culture medium. 7 After mixing Xc with A2780 cells, they were incubated overnight in a cell culture incubator for 12 hours.

[0028] Mitochondria were extracted from A2780 cells incubated with Xc using a mitochondrial extraction kit (BioYunTian, ​​catalog number: C3601). Mitochondria were lysed using 50 μL of mitochondrial lysis buffer from the kit. After centrifugation at 12,000 rpm, the supernatant was collected. Non-denaturing protein loading buffer (BioYunTian, ​​catalog number: P0016) was added to the supernatant and mixed thoroughly by pipetting.

[0029] Place the protein electrophoresis tank on ice, add pre-chilled electrophoresis buffer, and add 40-50 μL of extracted, non-denatured protein solution to each well. Set the voltage to 150V and electrophoresis for 3-4 hours. Stop electrophoresis when bromophenol blue just runs off the gel. Remove the protein gel and place it in a fluorescence spectrometer. Select the CY3 channel for imaging. Based on the imaging results... Figure 2 As shown, a fluorescent band appeared on the non-denaturing separating gels at 8%, 10%, 12%, and 15%, respectively. The protein strips at each fluorescent position were cut and stored in a -80°C freezer.

[0030] The protein gels after fluorescence imaging were subjected to silver staining. The silver staining kit was purchased from Thermo Fisher Scientific (catalog number: 24600). The experimental procedure was performed according to the kit's instructions. Results Figure 2 As shown.

[0031] A separate portion of the undenatured protein solution was subjected to SDS-PAGE (SDS polyacrylamide gel electrophoresis). The gels and electrophoresis buffer were purchased from Beyotime (catalog numbers: P0012A and P0561). The experimental conditions were the same as those for Native-PAGE electrophoresis. After electrophoresis, fluorescence imaging was performed. Results Figure 2 As shown.

[0032] 2. Collect the non-denatured protein tapes from three repeated experiments for proteomic analysis. Figure 2 As shown, each experiment used 4 tapes for a total of 4 samples, with three replicates totaling 12 samples. The protein profiles were analyzed by Zhongke New Life (Zhejiang) Biotechnology Co., Ltd., contract number: BZ-AS-20212807. The protein profile results were then used for Venn enrichment of mitochondrial proteins (proteins used for Venn analysis must be detected in all three replicates on tapes of the same concentration). Figure 3 and Figure 4 As shown, common proteins and their respective unique proteins appeared on separating gels at 8%, 10%, 12%, and 15%.

[0033] 3. Based on the principle of molecular sieves, the higher the frequency of a protein's appearance on the four gradient gels, the higher its likelihood of binding to the target compound (Xc). Therefore, five proteins appearing on 8%, 10%, 12%, and 15% gels, and one protein appearing on both 8% and 10% gels, were selected. These six proteins were then ligated onto a CM5 chip, and the affinity of Xc for these proteins was detected using a macromolecular interaction analyzer (BIACORE). The results showed that the molecular chaperone protein (heat shock protein family Dmember 1, HSPD1) and succinate dehydrogenase complex flavoprotein subunit A, SDHA, could bind to Xc. Figure 5 and Figure 6 As shown.

[0034] 4. SDS-PAGE experiments showed that Xc did not affect the protein expression level of SDHA (results). Figure 7 As shown in the figure, to further investigate the effect of Xc on the protein function of SDHA, the effect of Xc on the enzyme activity of SDHA was detected using an SDHA kit purchased from Solarbio Science & Technology Co., Ltd. (catalog number: bc0950). The experimental procedure was performed according to the kit's instructions. Results Figure 8 As shown.

[0035] III. Test Results After diluting Xc to 50 mM with DMSO, it turned brown at the bottom of the centrifuge tube. Xc was represented by a red octagon. The experimental diagram is summarized as follows: Figure 1 ; Mitochondria were extracted from cells after Xc treatment, and then different protein bands were formed on 8%, 10%, 12%, and 15% non-denaturing separating gels. The protein bands were then subjected to proteomic analysis. Figure 2 As shown; CY3 channel imaging showed that Xc and the target protein formed four bands on 8%, 10%, 12%, and 15% non-denaturing separating gels (indicated by red dashed boxes). The control group (CTRL) was not treated with Xc. The experiment was repeated three times, yielding the same results, indicating that the binding of Xc to the protein persists after Native-PAGE electrophoresis, and the presence of protein bands was confirmed by silver staining. However, when the same treatment was performed using SDS-PAGE electrophoresis, no bands were observed on the 8%, 10%, 12%, and 15% denaturing separating gels, indicating that SDS disrupts the binding of Xc to the protein. Proteomic analysis was performed on the four bands on the 8%, 10%, 12%, and 15% non-denaturing separating gels, and mitochondrial proteins were enriched for further analysis.

[0036] Figure 3 As shown, similar to the principle mentioned in this invention, the most types of proteins are found on 8% gels, followed by 10% and 12% gels, and the fewest on 15% gels.

[0037] Figure 4 As shown, common and unique proteins appeared on the gels at 8%, 10%, 12%, and 15%.

[0038] We selected five proteins that appeared on gels at 8%, 10%, 12%, and 15% concentrations for validation, and the results are as follows: like Figure 5 As shown, it was found that HSPD1 can bind to Xc, and the maximum dissociation constant of the binding is KD = 7.304E-5(M).

[0039] like Figure 6 As shown, we selected SDHA, a protein that appeared on both 8% and 10% gels, for validation. We found that SDHA can also bind to Xc, with a maximum dissociation constant KD = 5.744E-5(M). To further determine whether Xc can regulate SDHA function, we examined the protein expression level and protease activity of SDHA. like Figure 7 As shown, Xc at different concentrations does not affect the expression level of SDHA protein, however, Figure 8 As shown, Xc significantly inhibited the enzyme activity of SDHA at different concentrations.

[0040] In summary, this invention successfully isolated, screened, and identified the target proteins of Xc by combining non-denaturing gradient gels (Venn enrichment molecular sieves), subcellular localization of fluorescent compounds, and analysis of overlapping proteins on different gradient gels.

[0041] Example 3 Application tests of Venn enrichment molecular sieves: I. Test Plan The polysaccharide Gc (Gc is derived from the fucosylated glycosaminoglycan derivative described in patent CN102329397A) was modified with a CY3 fluorescent probe. Except for replacing Xc with Gc, the experimental procedure was the same as that in Example 2.

[0042] II. Experimental Procedure Except for Gc replacing Xc, the rest of the experimental procedures are the same as those in Example 2.

[0043] III. Test Results like Figure 9As shown, due to the large molecular weight of polysaccharides, only three bands were formed on the 8%, 10%, and 12% gels. Protein bands were then analyzed using proteomics. The highest concentration of Gc (highest brightness) was observed on the 12% gel.

[0044] Figure 10 As shown, similar to the principle mentioned in this invention, the most protein types are found on the 8% gel, followed by the 10% gel, and the fewest on the 12% gel. Common and unique proteins appear on the 8%, 10%, and 12% gels. Figure 10 As shown, 18 proteins that appeared at 8%, 10%, and 12% were selected to verify their binding to Gc.

[0045] Example 4 Application tests of Venn enrichment molecular sieves: I. Test Plan The long-chain fatty acid Lc was modified with a CY3 fluorescent probe. Except that Lc replaced Xc, the experimental procedure was the same as that in Example 2.

[0046] II. Experimental Procedure Except for Lc replacing Xc, the rest of the experimental procedures are the same as those in Example 2.

[0047] III. Test Results like Figure 11 As shown, Lc binds to proteins and forms three bands on 8%, 12%, and 15% gels. These protein bands were then subjected to proteomic analysis. The highest Lc concentration (highest brightness) was observed on the 15% gel.

[0048] Figure 12 As shown, similar to the principle mentioned in this invention, the most protein types are found on the 8% gel, followed by the 12% gel, and the least on the 15% gel. Common and unique proteins appear on the 8%, 12%, and 15% gels. Figure 12 As shown, 12 proteins that appeared at 8%, 12%, and 15% were selected to verify their binding with Lc.

[0049] Example 5 Application tests of Venn enrichment molecular sieves: I. Test Plan The mitochondrial protein Pc was modified using a CY3 fluorescent probe. Except for replacing Xc with Pc, the experimental protocol was the same as in Example 2. The highest Pc concentration (highest brightness) was observed on the 12% gel.

[0050] II. Experimental Procedure Except for Pc replacing Xc, the rest of the experimental procedures are the same as those in Example 2.

[0051] III. Test Results like Figure 13 As shown, after Pc binds to the protein, it forms four bands on gels at 8%, 10%, 12%, and 15% concentrations. The protein bands formed are then subjected to proteomic analysis.

[0052] Figure 14 As shown, similar to the principle mentioned in this invention, the most types of proteins are found on 8% gels, followed by 10% and 12% gels, and the fewest on 15% gels. Figure 14 As shown, 39 proteins that appeared at 8%, 10%, 12%, and 15% were selected to verify their binding with Pc.

[0053] Example 6 Application tests of Venn enrichment molecular sieves: I. Test Plan The coding region DNA sequence Dc of the mitochondrial genome was modified using a CY3 fluorescent probe. Except for replacing Xc with Dc, the experimental protocol was the same as in Example 2. The highest concentration (highest brightness) of Dc was observed on the 15% gel.

[0054] II. Experimental Procedure Except for replacing Xc with Dc, the rest of the experimental procedures are the same as those in Example 2.

[0055] III. Test Results like Figure 15 As shown, after Dc binds to the protein, it forms four bands on gels at 8%, 10%, 12%, and 15% concentrations. The protein bands formed are then subjected to proteomic analysis.

[0056] Figure 16 As shown, similar to the principle mentioned in this invention, the most types of proteins are found on 8% gels, followed by 10% and 12% gels, and the fewest on 15% gels. Figure 14 As shown, 30 proteins that appeared at 8%, 10%, 12%, and 15% were selected to verify their binding to Dc.

Claims

1. A method for screening proteins that bind to target compounds, characterized in that, The method uses Venn enriched molecular sieves, which are composed of non-denaturing separating gels with different gradients; The method includes the following steps: (a) The target compound is incubated with a biological sample under non-denaturing conditions, wherein the target compound is a small molecule compound Xc; or the target compound is a polysaccharide Gc, a long-chain fatty acid, a mitochondrial protein, or a coding region DNA sequence of the mitochondrial genome that still has biological activity after being modified with a fluorescent group. (b) Proteins were extracted under non-denaturing conditions and subjected to non-denaturing polyacrylamide gel electrophoresis using the Venn enrichment sieve. (c) Locating the protein bands formed by the target compound on the non-denaturing separating gels of different gradients by fluorescence imaging; (d) Cut the protein bands on each gradient gel and perform proteomic analysis; (e) Overlapping proteins that appear on the non-denaturing separating gels of different gradients are screened by Venn analysis and verified as candidate binding proteins of the target compound.

2. The method according to claim 1, characterized in that, The gradient of the non-denaturing separating gels with different gradients is 8%~15%.

3. The method according to claim 2, characterized in that, The gradients of the non-denaturing separating gels with different gradients are 8%, 10%, 12%, and 15%, respectively, from smallest to largest.

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