阅读说明：本技术 一种用于dpp-4活性检测的质谱探针及其制备方法和应用 (Mass spectrum probe for DPP-4 activity detection and preparation method and application thereof ) 是由 王毅 程翼宇 李振皓 张晓慧 于 2019-10-14 设计创作，主要内容包括：本发明公开了一种用于DPP-4活性检测的质谱探针及其应用。该质谱探针包括氨基酸序列为Glu-Pro-Phe-Lys的多肽及修饰在所述多肽的谷氨酸的哌嗪类化合物。本发明提供的质谱探针由可被DPP-4特异性识别的多肽Glu-Pro-Phe-Lys与高质谱响应的小分子哌嗪类化合物连接而成,不仅能被DPP-4特异性识别并酶切,同时具有极高的质谱响应,质谱检测准确度高,能够准确反应DPP-4的活性,适用于血清等复杂体系中酶活性的检测,也适用于从中药等复杂体系中筛选具有DPP-4抑制活性的化合物。(The invention discloses a mass spectrometry probe for DPP-4 activity detection and application thereof. The mass spectrometry probe comprises a polypeptide with an amino acid sequence of Glu-Pro-Phe-Lys and a piperazine compound for modifying glutamic acid of the polypeptide. The mass spectrum probe provided by the invention is formed by connecting the polypeptide Glu-Pro-Phe-Lys which can be specifically identified by DPP-4 and a micromolecule piperazine compound with high spectrum response, not only can be specifically identified and enzyme-digested by DPP-4, but also has extremely high mass spectrum response, has high mass spectrum detection accuracy, can accurately reflect the activity of DPP-4, is suitable for detecting the enzyme activity in complex systems such as serum and the like, and is also suitable for screening compounds with DPP-4 inhibitory activity from complex systems such as traditional Chinese medicines and the like.)

1. A mass spectrometry probe for DPP-4 activity detection is characterized by comprising a polypeptide with an amino acid sequence of Glu-Pro-Phe-Lys and a piperazine compound modified on glutamic acid of the polypeptide.

2. The mass spectrometry probe for DPP-4 activity detection according to claim 1, wherein the piperazine compound is 1- (2-pyrimidinyl) piperazine, 1- (4-pyridyl) piperazine or 1- (1-methyl-4-pyridyl) piperazine.

3. The mass spectrometry probe for the detection of DPP-4 activity according to claim 2, having the formula (i):

4. the method of preparing a mass spectrometry probe according to any one of claims 1 to 3, comprising: synthesizing the polypeptide with the amino acid sequence of Glu-Pro-Phe-Lys by a solid phase method, carrying out solid phase synthesis polypeptide reaction on the polypeptide and piperazine compounds, and purifying to obtain the mass spectrum probe.

5. The method of preparing a mass spectrometry probe of claim 4, wherein the polypeptide is prepared by: firstly, resin is swelled by dichloromethane, then 3 times molar excess of protected amino acid raw material is added, 5 times molar excess of N, N-diisopropylethylamine is added for reaction, and 20% piperidine-dimethylformamide solution is added for deprotection after the reaction is finished; repeating the steps to connect amino acid Lys, Phe, Pro and Glu in sequence to obtain the compound.

6. Use of a mass spectrometry probe according to any one of claims 1 to 3 for the detection of DPP-4 activity.

7. Use of a mass spectrometry probe according to any one of claims 1 to 3 in the preparation of a reagent for detecting DPP-4 activity in serum.

8. Use of a mass spectrometry probe according to any one of claims 1 to 3 in the screening of DPP-4 inhibitors.

Technical Field

The invention belongs to the field of drug screening and evaluation methods, and particularly relates to a mass spectrometry probe for DPP-4 activity detection, and a preparation method and application thereof.

Background

Incretins (incretins) are polypeptide hormones which are generated in the intestinal tract and have the function of promoting insulin secretion, and have the functions of not only stimulating insulin secretion, but also inhibiting postprandial glucagon secretion, delaying intestinal emptying, inhibiting appetite, enhancing insulin sensitivity and the like. Incretins include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), both of which exhibit advantages in promoting insulin release and regulating insulin levels extra-pancreatically. However, GLP-1 and GIP are quickly hydrolyzed by DPP-4 in vivo, and the hydrolysis products influence the exertion of physiological functions, so that the GLP-1 and the GIP cannot be well applied clinically.

DPP-4 is a serine peptidase, which specifically recognizes proline or alanine at the second position of the N-terminal of incretins, and cleaves 2 amino acids at the N-terminal to inactivate the incretins, thereby affecting the physiological action of the incretins and further affecting the development of diabetes. Research shows that DPP-4 activity has a close relation with the occurrence and development of diabetes, and the DPP-4 inhibitor can reduce the catalytic activity of DPP-4 and inhibit the hydrolysis of incretins, so that the concentration of the incretins is increased, and the effects of improving blood sugar and protecting beta cell functions are achieved.

Currently, commonly used methods for screening the DPP-4 inhibitor include a fluorescent substrate method using glycine-proline-7-amino-4-methylcoumarin as a substrate and a chromogenic substrate method using glycylproline p-nitroaniline as a substrate. Because these methods have high requirements on reaction time and sample treatment, repeated verification or mutual verification by other methods is required, and it is difficult to directly measure the DPP-4 activity.

In order to solve the above problems, the present inventors disclosed a polypeptide for DPP-4 detection and a fluorescent probe comprising the polypeptide in patent application No. cn201510507259. x. The fluorescent probe is formed by connecting a polypeptide for specifically recognizing DPP-4 with an aggregation-induced emission molecule, and the amino acid sequence of the polypeptide is as follows: glutamic acid-proline-phenylalanine-lysine (EPKF), aggregation-inducing luminescent molecules are linked to lysine in the polypeptide. The second position of the N end of the polypeptide contains proline, so that the polypeptide in the fluorescent probe can be specifically recognized by DPP-4, and the polypeptide is cut; therefore, the fluorescent probe basically has no fluorescence absorption, but when the N-terminal dipeptide is lost, obvious fluorescence absorption is generated, so that the DPP-4 activity can be specifically detected in real time.

In protein analysis, protein samples are often pretreated in order to improve the sensitivity and specificity of detection and to broaden the detection range. Chemical derivatization of peptide fragments is a commonly used sample pretreatment method in protein analysis, and the purpose of improving detection sensitivity is achieved by performing derivatization treatment on amino groups, sulfydryl groups or carboxyl groups of the peptide fragments and introducing micromolecule labels easy to ionize.

Disclosure of Invention

The invention aims to provide a mass spectrum probe with high spectral response, which is used as an enzyme substrate, is specifically identified by DPP-4, and detects the activity of DPP-4 by mass spectrometry of the amount of the probe or enzyme digestion product before and after enzyme digestion reaction.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a mass spectrometry probe for DPP-4 activity detection, comprising: polypeptide with an amino acid sequence of Glu-Pro-Phe-Lys and piperazine compound modified on glutamic acid of the polypeptide.

The mass spectrometry probe is formed by connecting a polypeptide which can be specifically recognized by DPP-4 and a small molecule with high spectral response. The amino acid sequence of the polypeptide is as follows: glutamic acid-proline-phenylalanine-lysine (SEQ ID NO.1), wherein the micromolecule with high spectral response is a piperazine compound, and-NH of the piperazine compound is condensed with carboxyl of glutamic acid on polypeptide chain.

The DPP-4 enzyme cutting site is an amido bond between proline and phenylalanine, so the enzyme cutting products of the mass spectrum probe are piperazine compounds-glutamic acid-proline and phenylalanine-lysine. Because the piperazine compound has strong mass spectrum response, the activity of DPP-4 can be detected by measuring the amount of the probe or enzyme digestion product before and after the reaction.

Preferably, the piperazine compound is 1- (2-pyrimidinyl) piperazine, 1- (4-pyridyl) piperazine or 1- (1-methyl-4-pyridyl) piperazine.

More preferably, the piperazine compound is 1- (2-pyrimidinyl) piperazine, and the structure of the mass spectrometry probe is shown as the following formula (I):

the invention also provides a preparation method of the mass spectrometry probe for DPP-4 activity detection, which comprises the following steps: synthesizing the polypeptide with the amino acid sequence of Glu-Pro-Phe-Lys by a solid phase method, carrying out solid phase synthesis polypeptide reaction on the polypeptide and piperazine compounds, and purifying to obtain the mass spectrum probe.

The polypeptide is prepared by the following method: firstly, resin is swelled by dichloromethane, then 3 times molar excess of protected amino acid raw material is added, 5 times molar excess of N, N-diisopropylethylamine is added for reaction, and 20% piperidine-dimethylformamide solution is added for deprotection after the reaction is finished; repeating the steps to connect amino acid Lys, Phe, Pro and Glu in sequence to obtain the compound.

In a second aspect, the invention provides an application of the mass spectrometry probe in DPP-4 activity detection. The invention also provides application of the mass spectrometry probe in preparing a reagent for detecting the activity of DPP-4 in serum. The invention also provides application of the mass spectrometry probe in DPP-4 inhibitor screening.

Compared with the prior art, the invention has the beneficial effects that:

the mass spectrum probe provided by the invention is formed by connecting the polypeptide Glu-Pro-Phe-Lys which can be specifically identified by DPP-4 and a micromolecule piperazine compound with high spectrum response, not only can be specifically identified and enzyme-digested by DPP-4, but also has extremely high mass spectrum response, has high mass spectrum detection accuracy, can accurately reflect the activity of DPP-4, is suitable for detecting the enzyme activity in complex systems such as serum and the like, and is also suitable for screening compounds with DPP-4 inhibitory activity from complex systems such as traditional Chinese medicines and the like.

Drawings

FIG. 1 is a flow chart of DPP-4 mass spectrometry probe for enzyme activity detection.

FIG. 2 is an HPLC chromatogram for purity analysis of a DPP-4 mass spectrometric probe.

FIG. 3 is a HPLC-QqQ-MS extraction ion flow chromatogram for DPP-4 mass spectrum probe structure confirmation, wherein the upper right drawing is a mass spectrum result chart.

FIG. 4 shows a DPP-4 mass spectrometric probe¹H nuclear magnetic resonance spectrogram.

FIG. 5 is a graph showing the detection sensitivity of DPP-4 mass spectrometry probe for DPP-4.

FIG. 6 is a diagram showing the result of examining the detection specificity of DPP-4 by a DPP-4 mass spectrum probe and other proteins.

FIG. 7 is a graph showing the dose-effect relationship between the concentration of an inhibitor and its effect on DPP-4 inhibition.

FIG. 8 is a graph showing the results of measurement of DPP-4 enzyme activity in serum.

Detailed Description

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