A method for screening antiplatelet active components based on platelet protein ultrafiltration affinity mass spectrometry

Abstract

The present invention provides a method for screening antiplatelet active components based on ultrafiltration affinity mass spectrometry of platelet proteins. First, a test solution containing the target compound is prepared; then, platelet protein solution is extracted from a blood sample; next, the test solution is mixed with the platelet protein solution and subjected to a co-incubation operation; subsequently, a polymer of platelet protein and potential affinity components in the test solution is obtained by elution through an ultrafiltration tube; then, the platelet protein is dissociated by an organic solvent to obtain a solution of potential active components; further, mass spectrometry detection is performed on the obtained control solution and potential active component solution; subsequently, one-dimensional statistical analysis is carried out for screening and identification of the active components. Finally, the present invention also selects representative components among the potential active components to verify through platelet aggregation experiments. The present invention can screen antiplatelet active components in complex systems such as traditional Chinese medicine with high throughput.

Description

Technical Field

[0001] The present invention belongs to the field of high-throughput screening, and particularly relates to a method for discovering antiplatelet active ingredients based on platelet protein ultrafiltration affinity mass spectrometry Background Art

[0002] Shexiang Baoxin Pills are derived from Suhexiang Pills recorded in Taiping Huimin Heji Jufang, and are a modern traditional Chinese medicine compound composed of seven traditional Chinese medicines. It has the effects of aromatic warming and dredging, supplementing qi and strengthening the heart. In recent years, large-scale evidence-based research on traditional Chinese medicines for coronary heart disease has shown that Shexiang Baoxin Pills are safe and effective in the treatment of coronary heart disease, and can significantly reduce the incidence of adverse cardiovascular events caused by antiplatelet drugs. Previous studies have also found that Shexiang Baoxin Pills can inhibit the expression of platelet activation-related proteins. Therefore, it is very likely that Shexiang Baoxin Pills achieve the therapeutic effect on coronary heart disease by inhibiting platelet activation. However, the specific antiplatelet active ingredients in Shexiang Baoxin Pills are not yet clear

[0003] Establishing a high-throughput screening method to find the antiplatelet active ingredients in Shexiang Baoxin Pills is of great significance for the research and development of new drugs for cardiovascular diseases. At present, conventional methods for discovering active ingredients of traditional Chinese medicines mainly rely on separation and purification and pharmacological models to separate and detect the activities of monomers. However, these methods are cumbersome to operate, have a long research cycle and low accuracy, resulting in low efficiency in discovering antiplatelet active ingredients. Ultrafiltration affinity mass spectrometry can provide a method for accurately and quickly identifying potential affinity ligands by identifying the interaction between drugs and biological macromolecules. It can obtain its affinity ligands from natural products through protein targets to screen the corresponding active ingredients. Most current antiplatelet drugs treat cardiovascular diseases by targeting platelet function receptors, and these targets are mainly located on the platelet membrane and inside. Previous studies have also screened antiplatelet active ingredients by extracting platelet membrane proteins to prepare biological chromatography. Therefore, perhaps we can also screen antiplatelet active ingredients in natural products by extracting platelet proteins for affinity mass spectrometry Summary of the Invention

[0004] In view of the above technical problems in the prior art, the present invention provides a method for screening active ingredients based on platelet protein ultrafiltration affinity mass spectrometry, and the method for screening active ingredients based on platelet protein ultrafiltration affinity mass spectrometry is to solve the problem of low efficiency in screening antiplatelet active ingredients in traditional Chinese medicines in the prior art

[0005] The present invention provides a method for screening antiplatelet active ingredients based on platelet protein ultrafiltration affinity mass spectrometry, comprising the following steps

[0006] S1. Prepare a test solution containing a target compound

[0007] S2. Extract platelet protein solution from the blood sample;

[0008] S3. Incubate the test solution from S1 with the platelet protein solution from S2;

[0009] S4. Use an ultrafiltration tube to elute the co-incubation solution in S3 to obtain a polymer of platelet protein and the platelet protein-affinity small molecule in the test solution;

[0010] S5. Dissociate the small molecule and platelet protein polymer in S4 to obtain a solution of potential active ingredients; S6. Use LC-MS to detect the dissociated small molecules in S5;

[0011] S7. Screen the mass spectrometry data obtained in S6 by one-dimensional statistical analysis of the data of the control group and the platelet protein affinity group to screen the small molecules in the test solution that are affinity to platelet protein, that is, potential antiplatelet active ingredients.

[0012] S8. Conduct an in vitro verification of the potential antiplatelet active ingredients in S7 through a platelet aggregation experiment.

[0013] Further, step S1 is specifically as follows:

[0014] Use a suitable extraction method to extract the components in the target sample as comprehensively as possible, evaporate the solvent to dryness, and re-dissolve with a buffer solution (25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM dithiothreitol, and 10 mM MgCl 2 ) to obtain a test solution for standby.

[0015] Further, step S2 is specifically as follows:

[0016] 1) Whole blood extraction: Anesthetize SD rats with sodium pentobarbital at a mass percentage concentration of 2%, take blood from the abdominal aorta of SD rats, and place the whole blood in a centrifuge tube containing ethylenediaminetetraacetic acid at a mass percentage concentration of 5%;

[0017] 2) Platelet-rich plasma extraction: Centrifuge the blood sample anticoagulated with ethylenediaminetetraacetic acid at a mass percentage concentration of 5% at room temperature. The upper layer after centrifugation is platelet-rich plasma, and the lower layer is precipitated white blood cells and red blood cells;

[0018] 3) Platelet extraction: Transfer the platelet-rich plasma to a new centrifuge tube, centrifuge, and discard the upper plasma to precipitate pure platelets;

[0019] 4) Platelet protein extraction: Resuspend the pure platelets in a buffer solution, add an appropriate amount of protease inhibitor phenylmethylsulfonyl fluoride at a mass percentage concentration of 1%, and after ultrasonic disruption, obtain a platelet protein suspension for standby.

[0020] Further, step S3 is specifically as follows:

[0021] Mix the platelet protein suspension and the test solution at a volume percentage of 1:1 and incubate at 37°C.

[0022] Further, step S4 is specifically as follows:

[0023] 1) Preparation of ultrafiltration tube: Use a 10 kDa ultrafiltration centrifugal tube. Add ultrapure water before use, with the water level completely covering the membrane, and pre-cool in an ice bath or refrigerator for 10 minutes;

[0024] 2) Adding the incubation solution to the ultrafiltration tube: Operate on ice, pour out the water, and add the above-mentioned S3 incubation solution to the 10 kDa ultrafiltration tube;

[0025] 3) Centrifugation and washing: Centrifuge the 10 kDa ultrafiltration centrifugal tube and the incubation solution at 4°C. After stopping centrifugation, wash the mixture with 50 mM ammonium acetate solution and then centrifuge;

[0026] Further, step S5 is specifically as follows:

[0027] Transfer the mixture of S4 to a new EP tube, extract the compound bound to the platelet protein using methanol, then centrifuge at 4°C, discard the protein precipitate, concentrate by drying with a nitrogen stream, and re-dissolve for testing.

[0028] Further, step S6 is specifically as follows:

[0029] Use high-resolution mass spectrometry to detect the test solution obtained in S5.

[0030] Further, step S7 is specifically as follows:

[0031] After performing one-dimensional statistical analysis using Progenesis QI analysis software, screen for differential ions between the control group and the affinity group, so as to obtain potential antiplatelet active ingredients:

[0032] Further, step S8 is specifically as follows:

[0033] In order to verify the antiplatelet activity of the screened potential ingredients, conduct an antiplatelet aggregation experiment to verify it.

[0034] The present invention uses a high-throughput screening method for antiplatelet active ingredients based on platelet protein ultrafiltration affinity mass spectrometry to screen for antiplatelet active ingredients in traditional Chinese medicines. Antiplatelet drugs usually bind to platelet surface proteins or specific proteins within platelets, interfering with their aggregation or activation processes, thereby achieving the purpose of inhibiting platelet function and reducing thrombus formation. Platelet aggregation is a key process in thrombus formation. Therefore, searching for small molecule drugs that can effectively interfere with platelet activity is an important direction for the development of antiplatelet drugs. The ultrafiltration affinity separation technique uses platelet proteins as the affinity phase and separates antiplatelet active ingredients from traditional Chinese medicine components by selectively adsorbing compounds that are affinity with platelet proteins. This process enables efficient screening of active ingredients through the binding characteristics between platelet proteins and candidate compounds. Combining with mass spectrometry technology, the traditional Chinese medicine components that bind to platelet proteins can be accurately identified. Mass spectrometry can provide molecular mass information and structural characteristics of each compound, helping researchers identify potential antiplatelet active ingredients. At the same time, mass spectrometry technology has high sensitivity and high resolution, can handle complex traditional Chinese medicine component libraries, and quickly screen out potentially active molecules. Therefore, the advantages of the high-throughput screening method for antiplatelet active ingredients based on platelet protein ultrafiltration affinity mass spectrometry enable it to effectively identify potential antiplatelet components in traditional Chinese medicines, providing new ideas and tools for the discovery of antiplatelet drugs.

[0035] The present invention has the following advantages over the prior art:

[0036] 1. The present invention can quickly screen and obtain potential antiplatelet active ingredients, improving the screening efficiency of active ingredients.

[0037] 2. The present invention is based on the ultrafiltration affinity mass spectrometry technology of platelet proteins and is applicable to the high-throughput screening of active ingredients in complex samples.

[0038] For better understanding and implementation, the present invention will be described in detail below with reference to the accompanying drawings. Description of the Drawings

[0039] Figure 1 Workflow diagram of the high-throughput screening method for antiplatelet active ingredients based on platelet protein ultrafiltration affinity mass spectrometry.

[0040] Figure 2 Method verification of the antiplatelet positive drug on platelet protein affinity mass spectrometry.

[0041] Figure 3 Screening of components in Shexiang Baoxin Pills that are affinity with platelet proteins.

[0042] Figure 4 Verification of platelet aggregation experiments of representative active ingredients. Detailed Implementation Modes

[0043] Example 1:

[0044] A method for screening antiplatelet active components based on ultrafiltration affinity mass spectrometry of platelet proteins.

[0045] Refer to Figure 1 , which is the workflow diagram of the high-throughput screening method for antiplatelet active components based on ultrafiltration affinity mass spectrometry of platelet proteins. First, extract the test solution of the target sample; then extract the platelet protein solution from the blood sample; then mix the test solution with the platelet protein solution and perform a co-incubation operation; subsequently, obtain the polymer of platelet protein and potential affinity components in the test solution by elution through an ultrafiltration tube; then dissociate the platelet protein with an organic solvent to obtain the solution of potential active components; further perform mass spectrometry detection on the obtained control solution and potential active component solution; then perform one-dimensional statistical analysis to screen and identify the active components. Finally, the present invention also selects several representative components from the potential active components for platelet aggregation experiment verification.

[0046] This example conducts high-throughput screening on the potential active components in Shexiang Baoxin Pills, specifically including the following steps:

[0047] (1) Preparation of buffer solution (25 mM Tris-HCl, 150 mM NaCl, 1 mM dithiothreitol, and 10 mM MgCl 2 ): Take 1 mL of 1 M Tris-HCl and dilute it 40 times with ultrapure water to 40 mL, add 348 mg of NaCl, 6.0 mg of dithiothreitol, and 83.4 mg of MgCl 2 6H 2 O;

[0048] Preparation of 150 mM ammonium acetate solution: Take 40 mL of ultrapure water and add 461.1 mg of ammonium acetate.

[0049] (2) Preparation of Shexiang Baoxin Pills solution: Take 4 Shexiang Baoxin Pills (each 25 mg), add 1 mL of methanol, ultrasonically extract for 0.5 h, then centrifuge at 13000 rpm for 10 min at 4 °C, and take 100 μL of the supernatant and dissolve it in 10 mL of buffer solution (25 mM Tris-HCl, 150 mM NaCl, 1 mM dithiothreitol, and 10 mM MgCl 2 ).

[0050] (3) Preparation of positive drug solution (the positive drug solution is the standard solutions of two commonly used antiplatelet drugs on the market, which are compounds known to be able to bind to platelet proteins. The positive drug solution is set here to verify the feasibility of the method with known binding compounds, and the control group solution is the buffer solution without platelet protein).

[0051] Prepare 0.02 mM stock solutions of ticagrelor and tirofiban positive drugs with 100% methanol respectively. Take 100 μL of each of the above positive drug stock solutions, mix them, and dilute with 10 mL of buffer (containing 25 mM Tris-HCl, 150 mM NaCl, 1 mM dithiothreitol, and 10 mM MgCl 2 ) to obtain the positive drug solution preparation solution.

[0052] (4) Platelet protein extraction: Take an appropriate amount of male SD rats, anesthetize them with sodium pentobarbital containing 2% (mass percentage concentration), collect blood from the abdominal aorta of male SD rats, and place the whole blood in a centrifuge tube containing 5% (mass percentage concentration) ethylenediaminetetraacetic acid. Centrifuge at 1200 rpm for 10 minutes. After centrifugation, take the upper platelet-rich plasma, and then centrifuge at 3500 rpm for 5 minutes. Discard the upper plasma, and take the precipitate as platelets. Resuspend the platelets in buffer (containing 25 mM Tris-HCl, 150 mM NaCl, 1 mM dithiothreitol, and 10 mM MgCl 2 ) and add an appropriate amount of 1% phenylmethylsulfonyl fluoride protease inhibitor. After ultrasonic disruption for 30 min, mix well to obtain the platelet protein solution.

[0053] (5) Incubation of test solution with platelet protein: Take 100 μL of platelet protein solution and 100 μL of positive drug solution respectively and incubate at 37 °C for 30 min for the method verification of the positive drug.

[0054] Take 6 portions of 100 μL of platelet protein solution and 100 μL of Shexiang Baoxin Pills solution respectively and incubate at 37 °C for 30 min as the affinity group samples. At the same time, take 6 portions of 100 μL of control buffer solution (containing 25 mM Tris-HCl, 150 mM NaCl, 1 mM dithiothreitol, and 10 mM MgCl 2 ) and 100 μL of Shexiang Baoxin Pills solution and incubate at 37 °C for 30 min as the control group samples.

[0055] (6) Ultrafiltration tube elution: Add the above incubation solution to a 0.5 mL 10 kDa ultrafiltration tube. Centrifuge the ultrafiltration centrifuge tube and the incubation solution at 13000 rpm at 4 °C for 10 min, wash the mixture five times with 200 μL of 150 mM ammonium acetate solution respectively, and then centrifuge to 100 μL.

[0056] (7) Dissociation of active ingredients: Transfer the above concentrated solution to new EP tubes respectively, extract the active ingredients bound to platelet proteins with 300 μL of methanol, then centrifuge at 13000 rpm at 4 °C for 10 min, and take the supernatant for testing.

[0057] (8) Chromatographic conditions: The chromatographic separation of the samples was performed using a Waters Acquity UPLC system. The chromatographic column was a Waters Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The column temperature was 40 °C. The mobile phase consisted of water (A) containing 0.1% formic acid by volume percentage concentration and acetonitrile (B). The flow rate was 0.3 mL / min. The gradient elution program was as follows: 0 - 1 min, 5% - 5% B; 1 - 13 min, 5% - 100% B; 13 - 15 min, 100% - 100% B; 15 - 15.1 min, 100% - 5% B; 15.1 - 20 min, 5% - 5% B; the injection volume was 2 μL.

[0058] (9) Mass spectrometry conditions: Mass spectrometry detection was performed on a SYNAPT G2-Si HDMS system. Data acquisition was carried out in both positive and negative ion modes using the fastDDA mode. The mass spectrometry conditions were set as follows: drying gas flow rate, 800 L / h; drying gas temperature, 400 °C; ion source temperature, 120 °C; capillary voltage, 2.5 kV (positive ion mode), 2.0 kV (negative ion mode); cone voltage, 40 V, source offset, 80 V; cone gas flow rate, 50 L / h; mass range, 50 - 1500 Da; MS scan rate, 0.2 s. The parameter settings for the fast DDA mode were as follows: low mass collision energy was 10 - 40 V, and high mass collision energy was 40 - 120 V. Real-time data calibration was performed using a 200 pg / μL leucine enkephalin solution. Data acquisition was obtained by MassLynx V4.1.

[0059] (10) Method validation: To verify the reliability of the platelet protein affinity mass spectrometry method, two antiplatelet positive drugs were incubated with platelet proteins by affinity, and whether the positive drugs were affinity with platelet proteins was verified by comparing the abundance differences with the control group.

[0060] (11) One-dimensional statistical analysis: The control group and affinity group data sets were subjected to peak alignment and peak extraction, and a characteristic ion quantification table containing all data grouping information, retention time, m / z, and peak area was exported. After the characteristic ion quantification table was corrected and normalized by StatTarget software, it was imported into the MetaboAnalyst platform, and potential platelet affinity active ions were screened according to the criteria of p < 0.05 and FC > 2, and they were identified.

[0061] (12) Verification of platelet aggregation experiment: The platelet aggregation experiments of two representative active ingredients (cinnamic acid - CA and taurocholic acid - TCA) were verified by turbidimetry using a platelet aggregometer. First, when preparing the TB solution, 504.6 mg of NaHCO 3, 4.032 g of NaCl, 108 mg of KCl, 203 mg of MgCl 2 ·6H 2 O, 32.76 mg of NaH 2 PO 4 ·H 2 O, 1192 mg of 4 - hydroxyethylpiperazineethanesulfonic acid was dissolved in 500 mL of ultrapure water. After preparation, the pH value was adjusted to 7.4 and stored at 4°C. Then, abdominal aortic blood of SD rats was collected. The collected blood was added with normal saline with a volume ratio of 1:1 and adenosine triphosphate diphosphatase with a volume ratio of 400:1, and centrifuged at 870 rpm for 20 minutes to obtain platelet - rich plasma. Subsequently, ethylenediaminetetraacetic acid was added and centrifuged at 1900 rpm for 10 minutes to obtain platelet - poor plasma. After discarding the supernatant, the platelets were deposited at the bottom of the aggregation tube. To prepare the washed platelet solution, the TB solution was mixed with glucose at a volume ratio of 1:1. Subsequently, the platelet aggregation was detected by turbidimetry using a platelet aggregometer. In the aggregometer pre - heated to 37°C, 300 μL of the washed platelet solution was added, and then 1.5 μL of the active ingredient solution was added. After incubation for 5 minutes, the aggregometer was adjusted to the baseline, and the agonist ADP was added, and the changes in the aggregation curve were recorded.

[0062] Figure 2 This example was for the method verification of platelet - positive drugs for platelet protein affinity mass spectrometry: Two positive drugs, ticagrelor and tirofiban, could significantly bind to platelet proteins, thus confirming the feasibility of the platelet protein affinity mass spectrometry - based screening technology in the high - throughput screening of anti - platelet active components in traditional Chinese medicine.

[0063] Figure 3 This example was for the screening of components in Shexiang Baoxin Pills that bind to platelet proteins: The control group and the affinity group data sets were subjected to one - dimensional statistical analysis, and components in Shexiang Baoxin Pills that bind to platelets were screened and identified with the criteria of p < 0.05 and FC > 2. Finally, 17 potential active components were screened out from Shexiang Baoxin Pills.

[0064]

[0065]

[0066] Figure 4 This example was for the verification of platelet aggregation experiments of representative active components: Two representative components, cinnamic acid (CA) and taurocholic acid (TCA), were selected from the 17 screened potential anti - platelet active components for platelet aggregation experiments. The results showed that within a certain concentration range, both cinnamic acid and taurocholic acid could inhibit platelet aggregation to a certain extent.

[0067] In summary, this embodiment uses a high-throughput screening method for antiplatelet active ingredients based on platelet protein ultrafiltration affinity mass spectrometry to screen the antiplatelet active ingredients in Shexiang Baoxin Pills. Antiplatelet drugs often achieve antiplatelet effects by binding to platelet proteins. Therefore, by screening the antiplatelet active ingredients in Shexiang Baoxin Pills that are affinity with platelet proteins, potential antiplatelet active ingredients can be quickly and high-throughput screened from the complex components of Shexiang Baoxin Pills. Finally, the platelet aggregation experiment can be used to verify the activity of the screened active ingredients.

[0068] The above-described embodiments merely represent one implementation mode of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. A method for screening antiplatelet active ingredients based on platelet protein ultrafiltration affinity mass spectrometry, characterized in that: The following steps are involved: S1. Prepare a test solution containing the target compound; S2, extracting platelet protein solution from the blood sample; S3, using S1 test solution and S2 platelet protein solution to incubate; S4, using an ultrafiltration tube to elute the co-incubation solution in S3, thereby obtaining a polymer of platelet protein and the platelet protein affinity small molecule in the test melt; S5, dissociating the small molecules in S4 from the platelet protein polymer to obtain a potential active ingredient solution; S6, using LC-MS to detect the small molecules after dissociation in S5; S7, screening the mass spectrometry data obtained in S6 by unidimensional statistical analysis of the control group and the platelet protein affinity group data, thereby screening small molecules in the test solution that have affinity with platelet proteins, that is, potential antiplatelet active components; S8, platelet aggregation experiment to verify the potential antiplatelet active components in S7 in vitro; Step S1 is specifically as follows: The extracted target sample is redissolved in a buffer solution containing 25 mM Tris-HCl, 150 mM NaCl, 1 mM dithiothreitol and 10 mM MgCl2, the components in the target sample are extracted, and the solvent is evaporated to obtain a test solution for use; Step S2 is specifically as follows: 1) Whole blood extraction: SD rats were anesthetized with 2% sodium pentobarbital, and blood was collected from the abdominal aorta of the SD rats. The whole blood was placed in a centrifuge tube containing 5% ethylenediaminetetraacetic acid; 2) Platelet-rich plasma extraction: The blood sample anticoagulated with 5% EDTA by mass concentration is centrifuged at room temperature. After centrifugation, the upper layer is platelet-rich plasma, and the lower layer is precipitated white blood cells and red blood cells; 3) Platelet extraction: transfer platelet-rich plasma into a new centrifuge tube, centrifuge, discard the upper plasma, and precipitate pure platelets; 4) Platelet protein extraction: resuspend the pure platelets in a buffer solution, add 1% by mass concentration of phenylmethylsulfonyl fluoride protease inhibitor, and after ultrasonic rupture, obtain a platelet protein suspension for use; Step S3 is as follows: Take the platelet protein suspension and mix it with the test solution in a volume percentage of 1:1, and incubate it at 37°C; Step S4 is specifically as follows: 1) Preparation of ultrafiltration tube: Use a 10 kDa ultrafiltration tube, add ultrapure water before use, and make sure the water completely passes through the membrane. Pre-cool in an ice bath or refrigerator for 10 minutes. 2) Add the incubation solution to the ultrafiltration tube: operate on ice, pour out the water, and add the above S3 incubation solution to the 10 kDa ultrafiltration tube; 3) Centrifugation and washing: Centrifuge the 10 kDa ultrafiltration centrifuge tube with the incubation solution at 4°C. After stopping the centrifugation, wash the mixture with 50 mM ammonium acetate solution and centrifuge again. Step S5 is specifically as follows: The S4 mixture was transferred to a new EP tube, and the compounds bound to platelet proteins were extracted using methanol, followed by centrifugation at 4°C, the protein precipitate was discarded, and the mixture was dried and concentrated using a nitrogen stream, and then re-dissolved for testing; Step S7 is specifically as follows: After unidimensional statistical analysis using Progenesis QI analysis software, the differential ions between the control group and the platelet protein affinity group were screened to obtain potential antiplatelet active components.

2. The method for screening antiplatelet active components based on platelet protein ultrafiltration affinity mass spectrometry according to claim 1, characterized in that: Step S6 is specifically as follows: The solution to be tested is obtained by high-resolution mass spectrometry.

3. The method for screening antiplatelet active components based on platelet protein ultrafiltration affinity mass spectrometry according to claim 1, characterized in that: Step S8 is specifically as follows: In order to verify the antiplatelet activity of the potential components screened, antiplatelet aggregation experiments were performed on them.

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