Conjoint analysis method for analyzing interaction between small molecule substance and targeted protein

Abstract

The invention belongs to the field of functional proteomics research, and particularly relates to a combined analysis method for analyzing interaction between a small molecule substance and a targeted protein, which is used for analyzing the small molecule substance and the protein by adopting LiP-MS (Limited Protein-Mass Spectrometry) technology and phosphorylated proteome combined analysis. According to the method, the optimized LiP-MS technology is adopted, the target protein can be directly recognized under the complex proteome background without enriching specific protein, and the problem that low-abundance protein is difficult to detect is solved; phosphorylated proteome abundance transformation protein is analyzed by combining high-resolution mass spectrometry, so that the recognition capability on a drug target and a shear variant thereof is enhanced, and the detection sensitivity is improved.

Description

Technical Field

[0001] The present invention belongs to the field of functional proteomics research, and particularly relates to a combined analysis method for analyzing the interaction between small molecule substances and target proteins. Background Art

[0002] Proteins are the core molecules that perform various functions in organisms, and abnormal expression and function of proteins are often closely related to the occurrence and development of diseases. Ki67, as an important cell proliferation marker, is widely used in tumor diagnosis and prognosis evaluation. In recent years, research on Ki67 has mainly focused on its expression pattern and clinical application in cancer, while research on its use as a drug target is still relatively scarce. Therefore, analyzing the mechanism of action of small molecule compounds such as Pomolic Acid (PA) on Ki67 is of great significance for tumor treatment and drug development.

[0003] Currently, the following several methods are mainly used to study the interaction between small molecule compounds and protein targets:

[0004] ① Principle of Thermal Stability Analysis (CETSA, Cellular Thermal Shift Assay): Based on the change in the thermal stability of the target protein after binding of the small molecule drug, protein detection is performed after heating at different temperatures

[0005] ② Principle of Protein-Small Molecule Interaction Screening Technology (DARTS, Drug Affinity Responsive Target Stability): The binding of a drug to a protein will change its sensitivity to proteases, and potential targets can be screened through proteolytic analysis.

[0006] ③ Principle of Affinity Purification-Mass Spectrometry (AP-MS): The target protein is enriched by an affinity purification method using immobilized drugs or ligands, and then the target is identified by mass spectrometry analysis.

[0007] ④ Principle of Chemical Proteomics: A small molecule probe is covalently bound to the target protein, and LC-MS / MS is combined for protein identification.

[0008] Although the above-mentioned technologies can analyze the interaction between small molecules and proteins to varying degrees, they have certain limitations in terms of high throughput, label-free, and applicability to low-abundance proteins. Especially for proteins like Ki67 with complex structures and controlled expression, traditional methods may be difficult to accurately analyze their targeted binding patterns.

[0009] Therefore, there is an urgent need for a more sensitive and efficient technical means to analyze the targeting effect of proteins such as Ki67. Summary of the Invention

[0010] The object of the present invention is to solve the problems existing in the analysis of the interaction between small molecule substances and their target proteins, such as insufficient sensitivity, limited scope of application, high false positive / false negative, inability to analyze conformational changes, and cumbersome experiments, and to provide a new method for analyzing the interaction between small molecule substances and target proteins.

[0011] A method for analyzing the combined interaction between small molecule substances and target proteins of the present invention adopts the following technical scheme:

[0012] A method for analyzing the interaction between small molecule substances and target proteins uses LiP-MS (Limited Proteolysis-Mass Spectrometry) technology to analyze the conformational changes of small molecule substances and proteins; uses phosphorylated proteomics to further analyze the functional proteins with expression changes to determine the final target protein.

[0013] LiP-MS (Limited Proteolysis-Mass Spectrometry) is a proteomics technology that has emerged in recent years and can be used to study protein-small molecule interactions. This technology utilizes the conformational changes of proteins to affect their sensitivity to proteases, thereby combining mass spectrometry analysis to identify potential binding sites. Screening potential targets through high-throughput phosphorylated proteomics can improve the efficiency of drug development. The combination of the two has not been applied to the analysis of the interaction between small molecule substances and target proteins, and the inventor first attempts this method. And using small molecule inhibitors with known targets can clearly determine their targets.

[0014] Preferably, the small molecule substance is pomolic acid (PA), and the target protein is Ki67.

[0015] The present invention also provides a method for jointly analyzing the targeting of pomolic acid to Ki67 based on LiP-MS technology and phosphorylated proteomics, including the following steps:

[0016] (1) Extract proteins and measure the protein concentration;

[0017] (2) Incubate the protein lysate with pomolic acid at room temperature, and add proteinase K for limited hydrolysis;

[0018] (3) Quench the reaction and inactivate it at high temperature to prepare a mass spectrometry sample;

[0019] (4) Analyze the hydrolyzed peptide segments by liquid chromatography-mass spectrometry (LC-MS), and combine quantitative proteomics and bioinformatics methods to analyze the binding sites of ecdysone and Ki67.

[0020] Preferably, in the LiP-MS, the mass spectrometry chromatographic conditions are as follows: Use an EASY-nanoLC 1200 system and an Exploris 480 mass spectrometer, and the chromatographic column is Acclaim PepMap TM C18, mobile phase A is 0.1% formic acid in water (0.1:99.9), and mobile phase B is ACN-H20-FA (80:20:0.1).

[0021] Preferably, in step (3), for heat inactivation, perform reduction, alkylation treatment in sequence, desalt and concentrate after enzymatic hydrolysis to obtain a mass spectrometry sample.

[0022] Preferably, the enzyme-to-protein ratio of proteinase K is 1:100, and the hydrolysis time is 4 minutes; the deoxycholate is a 2% sodium deoxycholate solution with a final concentration of 1%, and the heat inactivation condition is heating at 98°C for 15 minutes.

[0023] Preferably, the reduction treatment is: Add 100 mM DTT and react at 37°C for 30 minutes;

[0024] Preferably, the alkylation treatment is: Add 100 mM iodoacetamide (IAA) and incubate in the dark at room temperature for 20 minutes.

[0025] Preferably, add Lys-C and trypsin, and the enzyme-to-substrate ratios are 1:100 and 1:20 (volume ratio 1:100) respectively, and digest at 37°C and 800 rpm for 12 hours.

[0026] Preferably, the desalting is carried out using a solid-phase extraction column (Sep-Pak), and the specific steps include:

[0027] ① Activate the column with methanol;

[0028] ② Rinse with 50% acetonitrile-0.1% formic acid aqueous solution;

[0029] ③ Equilibrate with 0.1% formic acid solution and then load the sample;

[0030] ④ Desalt with 0.1% formic acid solution and elute the peptide segments with 50% acetonitrile-0.1% formic acid solution (in two portions).

[0031] Preferably, the acidification treatment conditions after enzymatic hydrolysis are: Add 98% formic acid to adjust the pH < 3, let stand at 4°C for 5 minutes, centrifuge at 16000 x g for 10 minutes and repeat centrifugation 3 times, filter to remove the precipitate, and then filter using a filter plate to obtain the clear liquid.

[0032] By implementing the above technical solutions, compared with the prior art, the present invention has the following advantages:

[0033] 1. Without relying on chemical modification or specific antibodies, the applicability is improved

[0034] Existing chemical proteomics methods rely on probe modification, while the LiP-MS of the present invention does not require chemical modification of the target acid and can be directly used for screening small molecule targets. Without using specific antibodies, the generality of the method is improved, and it is applicable to various small molecule-protein interaction studies.

[0035] 2. Reduce false positives / false negatives and improve the accuracy of target screening

[0036] By using limited proteolysis (LiP) combined with mass spectrometry, the cleavage pattern of proteases can be affected by protein conformational changes, thereby accurately identifying the binding sites of small molecules and proteins and reducing false positive results. Through quantitative proteomics analysis (such as TMT, SILAC and other labeling methods), the reliability of the data is further improved and the false negative rate is reduced.

[0037] 4. Directly analyze the conformational changes of the target acid after binding to Ki67

[0038] Traditional methods are difficult to directly observe the protein conformational changes caused by small molecule binding, while the LiP-MS technology of the present invention can reveal its mechanism of action through the changes in the secondary structure of proteins after small molecule binding. Combining phosphorylation site analysis provides more detailed binding mode and action site information.

[0039] 5. Simplify the experimental process and reduce the experimental period

[0040] By adopting an integrated mass spectrometry analysis process, compared with traditional CETSA or AP-MS methods, it reduces cumbersome steps such as protein enrichment and immunoassay, and directly analyzes through limited proteolysis and mass spectrometry identification. It is suitable for high-throughput screening, and for the analysis of quantitative proteomics, it greatly reduces the problem of false positive rate. It can analyze the effects of multiple small molecule compounds on potential target proteins in a short time and improve the research efficiency. Description of the Drawings

[0041] Figure 1 It is a flow chart for analyzing the interaction between small molecule substances and target proteins by the LiP-MS method using the present invention;

[0042] Figure 2 It is a flow chart of phosphoproteomics

[0043] Figure 3 It is a cluster analysis chart after differential treatment of drugs at multiple concentrations;

[0044] Figure 4 It is a schematic diagram of the pocket when the docking binding energy of euscaphic acid and Mki67 is the largest. Specific implementation mode

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0046] This specific embodiment is only an interpretation of the present invention, and it is not a limitation of the present invention. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.

[0047] Example 1

[0048] This embodiment provides a method for jointly analyzing the targeting of euscaphic acid to Ki67 based on LiP-MS technology and phosphoproteomics, including the following steps:

[0049] Pretreatment:

[0050] Extract the protein Ki67 from osteoclastic murine cells.

[0051] Take out the culture dish containing osteoclastic murine cells from the cell incubator. Carefully aspirate the culture medium, and gently wash the cells twice with pre-cooled PBS buffer. The purpose of each wash is to remove the serum and other impurity components in the culture medium to avoid interference with subsequent protein extraction. 2 mL of PBS buffer can be added each time, and then gently shake the culture plate to make the PBS buffer cover all the cells, and aspirate the PBS. Add 1 mL of self-prepared lysis buffer and (1:100) protease inhibitor. Use an ultrasonic crusher to break the cells (turn on for 3 s, stop for 2 s, power about 25%) until the liquid becomes clear and transparent. Centrifuge to collect the protein, and centrifuge the lysed cell sample at 4°C and 12,000 rpm for 15 minutes. The purpose of centrifugation is to remove impurities such as cell debris and unlysed organelles, and separate the protein supernatant from the impurities.

[0052] Adjust the concentration of the sample to 1 μg / μL according to the protein concentration determined by BCA. Take 100 μg of protein lysate, with 3 independent replicates each, and incubate with the drug at room temperature (RT) for 20 minutes. Set 4 concentration gradients of euscaphic acid (0, 25, 125, 625 μM). Add proteinase K (enzyme-to-protein ratio 1:100), and incubate for another 4 minutes. At this time, quench the activity of proteinase K with an equal volume of 2% deoxycholate (to a final concentration of 1%), and transfer the sample to a 98°C hot block for 15 minutes.

[0053] The sample was taken out from the heat source, reduced with 15 μL of 100 mM DTT at 37 °C for 30 min, and then incubated with 20 μL of 100 mM iodoacetamide (IAA) in the dark at room temperature for 20 min. Subsequently, DOC was diluted to 0.25% with four-fold 100 mM ammonium bicarbonate, pH was about 8, 1 μL of lysine endopeptidase (lys-C) (enzyme:substrate ratio 1:100) and 10 μL of trypsin were added, and digestion was carried out at 37 °C and 800 rpm for 12 h.

[0054] A certain amount of 98% formic acid was added to make the pH < 3, and it was left standing at 4 °C for 5 min. DOC precipitated upon encountering acid, and centrifugation was carried out at 16000 xg for 10 min. The centrifugation was repeated 3 times. Then filtration was carried out using a filter plate. After transferring the supernatant to a new EP tube, desalting of the digested solution was carried out using a Sep-Pak (waters) desalting column.

[0055] Desalting process: ① Activate the column with 1 mL of methanol; ② Wash with 1 mL of 50% acetonitrile + 0.1% formic acid aqueous solution; ③ Equilibrate twice with 1 mL of 0.1% formic acid aqueous solution; ④ Load the sample; ⑤ Elute three times with 1 mL of 0.1% formic acid aqueous solution; ⑥ Replace with a 2 mL recovery plate and elute twice with 500 μL of 50% acetonitrile + 0.1% formic acid aqueous solution.

[0056] Finally, the solvent of the eluted peptides was evaporated at 37 °C using a vacuum freeze concentration centrifuge.

[0057] Mass spectrometry analysis

[0058] Mass spectrometry chromatographic conditions:

[0059] Exploris 480 mass spectrometer of EASY-nanoLC 1200 (Thermo Fisher Scientific, MA, USA). Chromatography used Acclaim PepMap TM 100 C18 chromatographic column pre-column (75 μm × 2 cm, nanoViper 2Pk) and Acclaim PepMap TM RSLC chromatographic column (75 μm × 25 cm, nanoViper), mobile phase A was 0.1% formic acid in water (0.1:99.9), mobile phase B was ACN-H20-FA (80:20:0.1), gradient elution was adopted, flow rate was 150 nL / min, and injection volume was 2 μL.

[0060] The elution gradient of the liquid phase is shown in Table 1.

[0061] Table 1 Liquid phase elution gradient table

[0062]

[0063]

[0064] The experimental procedure for phosphoproteins is the same as the conventional experimental steps.

[0065] According to Figure 3 as shown, the abscissa group1, 2, 3, and 4 represent the control group, the administration group at 25 μM, the administration group at 125 μM, and the administration group at 625 μM in sequence. Through cluster analysis, structure-changing proteins with concentration dependence can be found, and then the molecular mechanism of its potential target can be analyzed based on the combination of proteins with concentration dependence and phosphorylated differential proteins.

[0066] Focusing on the common protein Mki67 through conformational changes and protein expression changes, in order to confirm the affinity between Mki67 and euscaphic acid, as well as the possible binding mode, this study used the method of molecular docking to predict the binding model between euscaphic acid and Mki67. Docking was performed 10 times, and the maximum molecular docking binding energy was -8.3 KJ / mol ( Figure 4 ), and the minimum docking binding energy was -5.3 KJ / mol; less than -5 KJ / mol indicates good docking binding energy.

[0067] The amino acid residues on the receptor protein that directly interact with the ligand in the pocket with the maximum binding energy during docking are as follows: Pocket 1: Chain A: GLN2595 ARG2622 LYS2625 GLU2626 LYS2627 GLU2633 SER2636 PHE2637 GLU2640 ARG2644 ARG2663 GLN2664 LEU2665 ARG2666 ILE2667 GLN2668 LEU2669 ILE2670 ASN2671 ASP2672 VAL2674 LYS2675 GLU2677 ALA2680 GLN2681 GLN2770 GLN2773 VAL2774 SER2776 PHE2777 ASP2921 THR2924 LYS2928 SER2944 ILE2945 ARG2949 ARG2952 SER2953.

[0068] Summary: The LiP-MS method was carefully optimized, and multiple drug concentrations were used to accurately screen for targets. Combining traditional proteomics to determine differentially quantified proteins based on protein abundance changes. Therefore, our invention can accurately screen for the potential target Ki67 of the ecdysteric acid drug. As a drug target, its strong binding effect was also demonstrated by molecular docking, and it was found that the drug is very likely to affect disease occurrence through cell proliferation and cell cycle-related pathways, thus providing strong evidence for new drug targets. It shows that the combined analysis of the optimized LiP-MS method and phosphoproteomics can more accurately find potential targets and discover new mechanisms of drug action.

Claims

1. A combined analysis method for analyzing the interaction between small molecule substances and target proteins, characterized in that Analysis of small molecules and proteins using LiP-MS technology and phosphoproteomics.

2. The method for analyzing the interaction between small molecule substances and target proteins according to claim 1, characterized in that, The small molecule is pomolic acid or its derivative, and the target protein is Ki67.

3. A method for jointly analyzing the targeting of pomolic acid to Ki67 based on LiP-MS technology and phosphoproteomics, characterized in that, It includes the following steps: (1) Extract proteins and measure protein concentration; (2) Incubate the protein lysate with pomolic acid at room temperature, and add proteinase K for limited hydrolysis; (3) Quench the reaction and inactivate at high temperature to prepare a mass spectrometry sample; (4) Analyze the hydrolyzed peptides using liquid chromatography-mass spectrometry (LC-MS) technology, and combine quantitative proteomics and bioinformatics methods to analyze the binding sites and conformational changes between pomolic acid and Ki67.

4. A method for analyzing the targeting of pomolic acid to Ki67 based on LiP-MS technology and phosphoproteomics according to claim 3, characterized in that, In the LiP-MS, the mass spectrometry chromatographic conditions are as follows: an EASY-nanoLC 1200 system and an Exploris 480 mass spectrometer are used, and the chromatographic column is Acclaim PepMap TM C18, mobile phase A is 0.1% formic acid in water, and mobile phase B is a mixed solution prepared by mixing ACN-H2O-FA in a volume ratio of 80:20:0.

1.

5. A method for analyzing the targeting of pomolic acid to Ki67 based on LiP-MS technology and phosphoproteomics according to claim 3, characterized in that, In step (3), the in-situ protein hydrolysis solution is used, proteinase K is used for peptide cleavage to obtain conformationally changed peptides, and after inactivation at high temperature, reduction treatment, alkylation treatment are carried out in sequence. After trypsin digestion, desalting and concentration are performed to obtain a mass spectrometry sample, thereby analyzing and obtaining conformationally changed proteins; the phosphoproteome obtains functional proteins with abundance changes.

6. A method for analyzing the targeting of pomolic acid to Ki67 based on LiP-MS technology and phosphoproteomics according to claim 3, characterized in that, The usage amount of proteinase K is proteinase K:protein = 1:100, and the hydrolysis time is 4 minutes; deoxycholate is used for quenching the reaction, and the deoxycholate is a 2% sodium deoxycholate solution with a final concentration of 1%.

7. A method for analyzing the targeting of pomolic acid to Ki67 based on LiP-MS technology and phosphoproteomics according to claim 5, characterized in that, The reduction treatment is: add 20 μL of 100 mM DTT and react at 37 °C for 30 minutes; the alkylation treatment is: add 15 μL of 100 mM iodoacetamide and incubate at room temperature in the dark for 20 minutes.

8. A method for analyzing the targeting of pomolic acid to Ki67 based on LiP-MS technology and phosphoproteomics according to claim 5, wherein, Lysine endopeptidase (Lys-C) and trypsin are added. The ratio of Lys-C enzyme to substrate is 1:100 respectively, and the volume ratio of trypsin is 1:100 (enzyme to substrate ratio 1:20). Digest at 37 °C and 800 rpm for 12 hours.

9. A method for analyzing the targeting of pomolic acid to Ki67 based on LiP-MS technology and phosphoproteomics according to claim 5, characterized in that, The desalting uses a solid-phase extraction column (Sep-Pak), and the specific steps include: ① Activate the column with methanol; ② Rinse with 50% acetonitrile-0.1% formic acid aqueous solution; ③ Equilibrate with 0.1% formic acid water and then load the sample; ④ Desalt with 0.1% formic acid water and elute the peptides with 50% acetonitrile-0.1% formic acid water (in two portions) in sequence.

10. A method for analyzing the targeting of pomolic acid to Ki67 based on LiP-MS technology and phosphoproteomics according to claim 5, characterized in that, The acidification treatment conditions after enzymatic digestion are: add 98% formic acid to adjust the pH < 3, add in small amounts and multiple times. Let it stand at 4 °C for 5 minutes, centrifuge at 16000 xg for 10 minutes and repeat 3 times, filter to remove the precipitate, and filter with a filter plate.

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