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A mass spectrometry probe for thrombin activity detection and its preparation method and application

A technology for activity detection and thrombin, which is applied in the field of mass spectrometry probes for thrombin activity detection and its preparation, can solve problems such as cumbersome procedures, limited applications, and susceptibility to interference, and achieve high accuracy, selectivity and exclusiveness of mass spectrometry detection High performance and sensitivity-enhancing effects

Active Publication Date: 2021-01-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is economical, simple, and easy to use, but has low repeatability and accuracy and is susceptible to interference, making it difficult to use for drug screening in complex systems
[0006] The anticoagulant method measures prothrombin time, thrombin time and thromboplastin time through a coagulometer, which requires experimental animals and the process is cumbersome
[0007] The chromogenic substrate method uses an optical detection method to measure the absorbance before and after the thrombin / substrate reaction. It has high specificity and is suitable for in vivo and in vitro detection. However, the chromogenic substrate method usually uses spectrophotometry, which has low sensitivity. And it is easily interfered by the background, and it is difficult to measure samples with strong absorption in the substrate absorption band, which greatly limits the application of this method in natural product screening

Method used

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  • A mass spectrometry probe for thrombin activity detection and its preparation method and application
  • A mass spectrometry probe for thrombin activity detection and its preparation method and application
  • A mass spectrometry probe for thrombin activity detection and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Synthesis of thrombin mass spectrometry probe

[0037] The structure of the thrombin mass spectrometry probe is D-phenylalanine-proline-arginine-β-alanine-1-(2-pyrimidinyl)piperazine (D-Phe-Pro-Arg-β- Ala-PP), synthesize peptides by solid-phase synthesis, and connect 1-(2-pyrimidinyl) piperazine (PP) groups with liquid-phase reactions, mainly including the following steps:

[0038] 1. Resin swelling

[0039] Weigh 2-Chlorotrityl Chloride Resin resin with a substitution degree of 0.4mmol / g, put the resin into a reaction tube, add dichloromethane, and shake for 30min.

[0040] 2. Take the first amino acid

[0041] Filter off the solvent with suction, add 3-fold molar excess of Fmoc-β-Ala-OH, then add 5-fold molar excess of diisopropylethylamine, finally add a small amount of dimethylformamide to dissolve, and shake for 1 h. Alternately wash 6 times with dimethylformamide and dichloromethane.

[0042] 3. Deprotection

[0043] Add 20% piperidine dimethylformamide solut...

Embodiment 2

[0053] Chemical characterization of thrombin mass spectrometry probe

[0054] The thrombin mass spectrometry probe was synthesized by the method described in Example 1, and the purity of the probe was analyzed by HPLC. Analysis conditions are: Jielun 1200HPLC chromatographic system, equipped with variable wavelength detector (VWD), detection wavelength 214nm; Chromatographic column, Kromasil C18 (4.6mm * 150mm, 5 μm); Mobile phase is 0.1% trifluoroacetic acid-water (A ) and 0.1% trifluoroacetic acid-acetonitrile (B); flow rate 1.0 mL / min; isocratic elution, 0-25 min, 5-70% B; injection volume, 10 μL.

[0055] After dissolving the thrombin mass spectrometry probe in pure water, centrifuge at 10,000 rpm for 5 minutes, and inject the sample. HPLC chromatogram as figure 1 shown. It can be seen from the figure that the synthesized thrombin mass spectrometry probe is of high purity, with a relative peak area ratio of >95%, which can be used for subsequent experiments and research...

Embodiment 3

[0059] Application of Thrombin Mass Spectrometry Probe in Detection of Thrombin Activity

[0060] A pH=8.3, 10 mM Tris-HCl was used as a buffer. Take 20 μL each of the thrombin mass spectrometry probe and different concentrations of thrombin solutions, and 160 μL of buffer solution, and place them in a 1.5 mL centrifuge tube, so that the final concentration of the thrombin mass spectrometry probe is 0.02 mM, and the final concentration of thrombin is 0.01- 0.2U / mL, incubate at 37°C for 2h. After the reaction was completed, 400 μL of methanol was added to terminate the reaction. The solution was vortexed and centrifuged, analyzed by the HPLC-IT / MS method described in Example 2, and the peak area difference of the thrombin mass spectrometry probe before and after the reaction reflected the thrombin activity, and the results were as follows image 3 shown. It can be seen from the figure that within this concentration range, there is a good linear relationship between the conce...

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Abstract

The invention discloses a mass spectrometry probe for thrombin activity detection, a method for preparing the mass spectrometry probe and application thereof, and belongs to the field of medicine screening and evaluation. The mass spectrometry probe comprises polypeptides and piperazines compounds. Amino acid sequences of the polypeptides are Phe-Pro-Arg-beta-Ala, and the piperazines compounds aremodified on beta-alanine of the polypeptides. The mass spectrometry probe, the method and the application have the advantages that the polypeptides Phe-Pro-Arg-beta-Ala capable of being specificallyrecognized by thrombin are linked with the small-molecular piperazines compounds with high mass spectrometry response to obtain the mass spectrometry probe, accordingly, the mass spectrometry probe can be specifically recognized by the thrombin, enzyme digestion can be carried out on the mass spectrometry probe by the thrombin, the mass spectrometry probe is extremely high in mass spectrometry response and mass spectrometry detection accuracy, the activity of the thrombin or the inhibitory activity of thrombin inhibitors can be accurately reflected by the mass spectrometry probe, and the massspectrometry probe is quite applicable to screening compounds with thrombin inhibitory activity from complicated systems such as traditional Chinese medicines.

Description

technical field [0001] The invention relates to the field of drug screening and evaluation, in particular to a mass spectrometry probe used for thrombin activity detection and its preparation method and application. Background technique [0002] Thrombin belongs to the serine protease family, plays an important role in the body's blood coagulation system, and participates in many key links in the blood coagulation process. Thrombin can promote the conversion of soluble fibrinogen in plasma into insoluble fibrin, activate multiple coagulation factors at the same time, and form thrombus. Thrombosis is closely related to many cardiovascular diseases, such as atherosclerosis, myocardial infarction and coronary heart disease. Therefore, thrombin is one of the important targets for the treatment of cardiovascular diseases. Direct thrombin inhibitors (direct thrombin inhibitors) have received great attention from drug developers because they can inhibit thrombin without cofactors....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K5/107C07K1/107C07K1/06C07K1/04C12Q1/56
CPCC07K5/1016C12Q1/56
Inventor 程翼宇李振皓
Owner ZHEJIANG UNIV
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