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Mass spectrometry probe for detecting activity of angiotensin converting enzyme and application thereof

An angiotensin and activity detection technology, applied in the field of drug screening and evaluation, can solve the problems of complex natural product components, difficult natural product screening, and low sensitivity, and achieve high selectivity and specificity, improved sensitivity, and high mass spectrometry response Effect

Active Publication Date: 2018-10-16
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of these methods require sample processing through multiple steps, cumbersome operation and low sensitivity
In addition, the detection principles of these methods are basically optical detection, which is easily disturbed by the background.
The composition of natural products is complex, and there may be strong absorption in the detection band of the substrate, so these methods are difficult to be directly applied to the screening of natural products

Method used

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  • Mass spectrometry probe for detecting activity of angiotensin converting enzyme and application thereof
  • Mass spectrometry probe for detecting activity of angiotensin converting enzyme and application thereof
  • Mass spectrometry probe for detecting activity of angiotensin converting enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Synthesis of ACE mass spectrometry probe

[0047]The structure of the ACE probe is 1-(2-pyrimidinyl)piperazine-aspartic acid-serine-aspartic acid-lysine-proline (PP-Asp-Ser-Asp-Lys-Pro), Synthesized by solid-phase synthesis, mainly comprising the following steps:

[0048] 1. Resin swelling

[0049] Weigh 2-Chlorotrityl Chloride Resin resin with a degree of substitution of 0.4 mmol / g, put the resin into a reaction tube, add dichloromethane (DCM), and shake for 30 minutes.

[0050] 2. Take the first amino acid

[0051] Filter off the solvent, add a 3-fold molar excess of Fmoc-L-Pro-OH, then add a 5-fold molar excess of diisopropylethylamine (DIEA), and finally add a small amount of dimethylformamide (DMF) to dissolve, shake 1h. Alternately wash 6 times with DMF and DCM.

[0052] 3. Deprotection

[0053] Add 20% piperidine DMF solution, react for 5 min, remove the solvent, add 20% piperidine DMF solution again, react for 15 min. After the reaction, wash twice with D...

Embodiment 2

[0061] Chemical Characterization of ACE Mass Spectrometry Probes

[0062] The ACE 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.

[0063] After dissolving the ACE mass spectrometer probe in pure water, centrifuge at 10,000 rpm for 5 minutes, and inject the sample. HPLC chromatogram as figure 1 shown.

[0064] It can be seen from the figure that the synthesized ACE mass spectrometry probe has a high purity and a relative peak area ratio of >95%, which can be used for subsequent experiments and research.

[0065] ...

Embodiment 3

[0068] Application of ACE mass spectrometry probe in the detection of ACE activity

[0069] A pH=8.3, 10 mM Tris-HCl was used as a buffer. Take 20 μL of ACE mass spectrometry probe and ACE solutions of different concentrations, and 160 μL of buffer solution, and put them in a 1.5 mL centrifuge tube, so that the final concentration of ACE mass spectrometry probe is 0.13 mM, and the final concentration of ACE is 0.005—0.06 U / mL , and incubated 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, and analyzed by the HPLC-IT / MS method described in Example 2, and the ACE mass spectrometer probe enzymatic hydrolysis product 1-(2-pyrimidinyl)piperazine-aspartic acid-serine- The amount of aspartic acid reflects the ACE activity, the results are as follows image 3 shown.

[0070] It can be seen from the figure that within this concentration range, there is a good linear relationship bet...

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Abstract

The invention discloses a mass spectrometry probe for detecting the activity of an angiotensin converting enzyme and application thereof, and belongs to the technical field of medicine screening and evaluation. The mass spectrometry probe comprises polypeptide having the amino acid sequence of Asp-Ser-Asp-Lys-Pro and a piperazine compound modified on aspartic acid at the N terminal of the polypeptide. The mass spectrometry probe provided by the invention is formed by connecting the polypeptide Asp-Ser-Asp-Lys-Pro capable of being specifically recognized by the angiotensin-converting enzyme, and a small molecular piperazine compound with high mass spectrometry response, not only can be specifically recognized and digested by the angiotensin converting enzyme, but also has very high mass spectrometry response and high mass spectrometry detection accuracy, can accurately reflect the activity of the angiotensin converting enzyme or the inhibitory activity of an angiotensin converting enzyme inhibitor, and is also very suitable for screening compounds having the angiotensin converting enzyme inhibiting activity from complex systems such as traditional Chinese medicines.

Description

technical field [0001] The invention relates to the technical field of drug screening and evaluation, in particular to a mass spectrometry probe for detecting angiotensin-converting enzyme activity and its application. Background technique [0002] Angiotensin-Converting Enzyme (ACE) is a zinc-dependent membrane-bound exopeptidase widely distributed in mammalian tissues. ACE can catalyze the conversion of angiotensin I to angiotensin II through the renin-angiotensin system (Renin-angiotensin system, RAS) and kallikrein-kinin system (Kallikrein-kinin system, KKS). Kinins are inactivated, leading to an increase in blood pressure. In addition, these two systems also play important regulatory roles in electrolyte and fluid balance, cardiovascular system development and functional remodeling. Therefore, ACE has become an important target for the treatment of diseases such as hypertension, acute myocardial infarction and heart failure. [0003] ACE inhibitors, such as captopril...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/04C07K1/06C07K1/107G01N33/68G01N33/573
CPCC07K7/06C07K19/00G01N33/573G01N33/6848G01N2410/00G01N2500/00
Inventor 程翼宇李振皓
Owner ZHEJIANG UNIV
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