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Mass spectrum probe used for DPP-4 activity detection, and preparation method and application thereof

A DPP-4 and activity detection technology, applied in the field of mass spectrometry probes for DPP-4 activity detection and their preparation, can solve problems such as difficult DPP-4 activity, and achieve the effects of high mass spectrometry response and high mass spectrometry detection accuracy

Inactive Publication Date: 2020-01-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since these methods have high requirements on reaction time and sample processing, repeated verification or mutual verification by other methods is required, and it is difficult to directly measure DPP-4 activity.

Method used

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  • Mass spectrum probe used for DPP-4 activity detection, and preparation method and application thereof
  • Mass spectrum probe used for DPP-4 activity detection, and preparation method and application thereof
  • Mass spectrum probe used for DPP-4 activity detection, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the synthesis of DPP-4 mass spectrometry probe

[0030] The synthesis method of the DPP-4 mass spectrometry probe of the present embodiment mainly comprises the following steps:

[0031] 1. Resin swelling

[0032] Weigh 0.6g of 2-Chlorotrityl Chloride Resin resin with a substitution degree of 0.4mmol / g, put the resin into a reaction tube, add dichloromethane (DCM) (15ml / g), and shake for 30min.

[0033] 2. Take the first amino acid

[0034] Filter off the solvent through a sand core, add a 3-fold molar excess of Fmoc-L-Lys(Boc)-OH amino acid, then add a 5-fold molar excess of N,N-diisopropylethylamine (DIEA), and finally add a small amount Dimethylformamide (DMF) was dissolved, shaken for 1 h, and finally washed 6 times alternately with DMF and DCM.

[0035] 3. Deprotection

[0036] Add 15ml 20% piperidine DMF solution (15ml / g), react for 5min, remove the solvent, add 15ml 20% piperidine DMF solution (15ml / g), react for 15min.

[0037] 4. Detection

...

Embodiment 2

[0053] Example 2: Chemical Characterization of DPP-4 Mass Spectrometry Probe

[0054] The DPP-4 mass spectrometer probe was synthesized by the method described in Example 1, and the purity of the probe was analyzed by HPLC. Analysis conditions are: Agilent 1260HPLC chromatographic system, equipped with variable wavelength detector (VWD), detection wavelength 214nm; chromatographic column, Agilent Zorbax SB-C 18 (4.6mm×100mm, 1.8μm); mobile phase is 0.05% formic acid-water (A) and 0.05% formic acid-acetonitrile (B); flow rate 0.4mL / min; gradient elution: 0-5min, 1%B; 5 -40min, 1-30%B; 40-45min, 30-100%B; 40-45min, 100%B; injection volume 10μL; column temperature 30°C.

[0055] After dissolving the DPP-4 mass spectrometry probe with pure water, centrifuge at 10000rpm for 10 minutes and then take the supernatant for sample analysis. The obtained HPLC chromatogram is as follows figure 2 shown.

[0056] Depend on figure 2 It can be seen that the DPP-4 mass spectrometry probe ...

Embodiment 3

[0059] Example 3: DPP-4 Mass Spectrometry Probe Detection Sensitivity of DPP-4

[0060] Take 5 μL mass spectrometry probe PP-DAPK (final concentration about 16 μM), different concentrations of DPP-4 (final concentrations are 500, 400, 250, 125, 50, 25, 10, 5, 2.5, 1.25, 0.5, 0.25, 0.05 μg / L), made up to 100 μL with 50 mM Tris-HCl pH 7.5, incubated at 37°C for 30 min, then added 200 μL methanol to stop the reaction, the solution was vortexed, centrifuged, and the supernatant was taken for injection analysis. The analysis conditions are: Agilent 1200HPLC chromatographic system, Api 4000 triple quadrupole mass in series (HPLC-QqQ-MS); ESI ion source; positive ion mode; MRM mode; source voltage, 4500V; source temperature 500 ° C; chromatographic column, Zorbax SB C18 (4.6mm×100mm, 1.8μm); the mobile phase is 0.05% formic acid-water (A) and 0.05% formic acid-acetonitrile (B); the flow rate is 0.4mL / min; the elution gradient is 0-1min, 1%B ;1-7min, 1-70%B; 7-7.5min, 70-99%B; 7.5-11...

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Abstract

The invention discloses a mass spectrum probe used for DPP-4 activity detection as well as a preparation method and application thereof. The mass spectrum probe used for DPP-4 activity detection comprises a polypeptide having an amino acid sequence of Glu-Pro-Phe-Lys, and a piperazine compound modified on glutamic acids of the polypeptide. Being composed of the polypeptide having the amino acid sequence of Glu-Pro-Phe-Lys, which can be specifically recognized by DPP-4, in connection with the small-molecular piperazine compound with high mass spectrum response, the mass spectrum probe providedby the invention can be specifically recognized and digested by the DPP-4; and moreover, the mass spectrum probe has very high mass spectrum response rate, and is high in mass spectrum accuracy and capable of accurately reflecting DPP-4 activity. Therefore, the mass spectrum probe is suitable for detection of enzyme activity in complex systems, such as serum and the like; and moreover, the mass spectrum probe is also suitable for screening of compounds with DPP-4 inhibitory activity from complex systems, such as traditional Chinese medicines and the like..

Description

technical field [0001] The invention belongs to the field of drug screening and evaluation methods, and in particular relates to a mass spectrometry probe used for DPP-4 activity detection and its preparation method and application. Background technique [0002] Incretin is a kind of polypeptide hormone produced in the intestinal tract with the effect of stimulating insulin secretion. It can not only stimulate insulin secretion, but also inhibit postprandial glucagon secretion, delay intestinal emptying, suppress appetite, Enhance insulin sensitivity and other effects. Incretins include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), both of which show many advantages in promoting insulin release and regulating insulin levels outside the pancreas. However, GLP-1 and GIP are quickly hydrolyzed by DPP-4 in vivo, and the hydrolyzed products affect their physiological functions, making them unable to be well applied clinically. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/113C07K1/04C07K1/06C07K1/107C12Q1/37G01N33/68
CPCC07K5/1021C12Q1/37G01N33/6848
Inventor 王毅程翼宇李振皓张晓慧
Owner ZHEJIANG UNIV
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