Antelope horn characteristic peptide fragment and detection method thereof

Abstract

The invention discloses an antelope horn characteristic peptide fragment and a detection method thereof, the antelope horn characteristic peptide with the sequence of Ile-Ser-Pro-Gly-Tyr-Ser-Val-Thr-Arg is screened through a large number of experiments, and the antelope horn characteristic peptide has high specificity and can be used for quality control of antelope horn medicinal materials and drugs containing the antelope horn medicinal materials. According to the detection method of the antelope horn characteristic peptide, the optimal chromatographic condition and mass spectrum condition are screened out through a large number of experiments, the whole method is easy to operate and accurate in judgment, and the antelope horn characteristic peptide can be accurately distinguished. The antelope horn characteristic peptide and the detection method thereof provided by the invention are beneficial to the quality control of antelope horn medicines, and have important significance for ensuring the quality of antelope horn-containing medicines.

Description

Technical Field

[0001] The invention relates to a detection and identification method for animal drugs, and in particular to a characteristic peptide segment of antelope horn and a detection method thereof. Background Art

[0002] Antelope horn Saigae Tataricae Cornu is the horn of Saiga tatarica Linnaeus, a bovine animal. It was first recorded in Shennong's Herbal Classics, and was called sheep, and commonly known as antelope. Its effects are: cold in nature, salty in taste, calming the liver and calming wind, clearing heat and calming convulsions, and detoxification; it is usually used for various febrile diseases, such as high fever headache, coma, delirium, mania, stroke, convulsions and rashes, etc. It is an important variety in clinical Chinese medicine and Chinese medicine pharmaceutical industry. Modern pharmacological studies have shown that antelope horn has good effects in sedation and hypnosis, anticonvulsant, analgesia, anti-epileptic, anti-inflammatory, antipyretic, anti-pathogenic microorganisms, anti-thrombosis, changing vascular permeability, anti-hypertension, antitussive and expectorant, and enhancing immunity. Clinical Chinese medicine and Chinese medicine pharmaceutical industry use antelope horn in Chinese patent medicine: Compound Antelope Horn Antihypertensive Tablets, Antelope Qingfei Pills, Antelope Xiaoyan Pills, etc., which have good therapeutic effects in clinical applications. Research on the material basis of antelope horns shows that proteins, peptides, amino acids, sterols, nucleosides, inorganic elements, etc. are its main components. The quality standards of antelope horns are not perfect. There is a lack of specific identification and content determination methods for antelope horn medicinal materials, concentrated powders, and prescription preparations containing antelope horns. There is a phenomenon of mixed sales with other horns or hoofs in the medicinal material market. Studies have shown that DNA barcoding technology based on COI sequences can be used to identify counterfeit antelope horn medicinal materials, but the high-temperature extraction process of traditional Chinese medicine will affect the integrity of DNA, and thus affect the accuracy of DNA barcode identification. Compared with DNA molecules, the primary sequence information of protein and peptide components is better preserved under high-temperature extraction conditions. Protein components such as type I keratin (Keratin, type I), type II keratin (Keratin, type II), and type I collagen (Type I Collagen) are important material bases for horn animal medicines such as antelope horns, buffalo horns, and goat horns. The present invention detects the components of antelope horn by detecting a characteristic peptide of antelope horn. The method provides a reference and basis for the quality research of antelope horn, and is conducive to further improvement of the quality standards of antelope horn and its products. Summary of the invention

[0003] The main purpose of the present invention is to provide a method for detecting antelope horn characteristic peptides in view of the above-mentioned problems and shortcomings. The antelope horn characteristic peptides provided by the present invention have strong specificity. The provided method is simple to operate and has high sensitivity, and can accurately identify whether the drug contains antelope horn ingredients, thereby providing a scientific method for ensuring the quality of antelope horns and their products.

[0004] Technical solution: In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0005] A characteristic peptide segment of antelope horn, the sequence of which is shown in Sequence Table 1:

[0006] Characteristic peptide: Ile-Ser-Pro-Gly-Tyr-Ser-Val-Thr-Arg.

[0007] A method for detecting characteristic peptide segments of antelope horns comprises the following steps:

[0008] (1) preparing a reference solution using the characteristic peptide fragments of antelope horn;

[0009] (2) After digesting the horn animal drug sample to be tested with trypsin, the enzymatic hydrolyzate and the mixed reference solution of the antelope horn characteristic peptide fragment in step (1) are injected into a liquid chromatography-mass spectrometer, and the antelope horn characteristic peptide fragment is used as a reference and the detection is performed in a multiple reaction monitoring mode; if an antelope horn characteristic peptide fragment ion pair is detected, and the retention time of the ion is consistent with that of the antelope horn characteristic peptide fragment reference substance, and its daughter ion is consistent with that of the reference substance, then the sample to be tested contains antelope horn components.

[0010] As a preferred embodiment, the above-mentioned method for detecting the characteristic peptide of antelope horn, the enzyme cleavage method is carried out as follows: take 10 mg of the horn animal drug sample to be detected, add 1 ml of protein lysis solution, homogenize 3 times, centrifuge, take the supernatant, add 0.2MIAA solution, place in dark, add cold acetone solution, mix well, refrigerate, and precipitate overnight;

[0011] After centrifugation of the acetone precipitate sample, discard the supernatant, wash the precipitate once with cold acetone, evaporate the precipitate, add urea solution to dissolve it, shake it thoroughly, add Tris-HCl buffer after the precipitate is completely dissolved, add trypsin to each horn sample respectively, and incubate in a constant temperature water bath to obtain the product.

[0012] As a preferred embodiment, the above-mentioned method for detecting the characteristic peptide of antelope horn, the enzyme cleavage method is carried out as follows: take 10 mg of the sample to be detected, add 1 ml of protein lysis solution (including 4% sodium dodecyl sulfate SDS, 20 mM dithiothreitol DTT, 50 mM Tris-HCl, pH = 8.8); add grinding beads, homogenize 3 times, each time for 60 seconds, centrifuge at 12000 rpm for 20 minutes, BCA determine the protein content, take 1000 μg of protein, add 25 μl of 0.2M IAA solution, place it away from light for 30 minutes, add 5.5V 样品 80% cold acetone solution, mix thoroughly and freeze at -20℃ for precipitation overnight;

[0013] Take the acetone precipitate sample, centrifuge it at 12000rpm for 20min, discard the supernatant, wash the precipitate twice with 80% cold acetone, evaporate the precipitate, add 200μl 8M urea solution to dissolve it, shake it thoroughly, add 1400μl Tris-HCl buffer after the precipitate is completely dissolved to reduce the urea concentration to below 1M, add 40μl of 0.1μg / μl trypsin to each horn sample, and incubate in a constant temperature water bath at 37℃ for 12h.

[0014] As a preferred embodiment, the detection method of the characteristic peptide of antelope horn described above, the liquid phase conditions for detection by liquid chromatography-mass spectrometry are as follows: the chromatographic column is a 1.8μm Waters T3 column with a specification of 2.1μm×100mm, the sample volume is 10μl, the flow rate is 0.3ml / min, the mobile phase A is 0.1% formic acid, the mobile phase B is acetonitrile, 0-5min, 5-20% B linear gradient elution, 5-8min, 20-95% B linear gradient elution, 8-9min, 95% B isocratic elution; a triple quadrupole mass spectrometer is used, and the mass spectrometry parameters are: ion source temperature 500°C; ionization voltage 5500V; desolvation temperature 500°C; ion source gas 1, 60psi; ion source gas 2, 60psi.

[0015] The ion pairs in MRM mode were: m / z 490.3 doubly charged→779.4, DP=40, CE=20.

[0016] As a preferred embodiment, in the above-mentioned method for detecting the characteristic peptide of antelope horn, the horn animal drugs include antelope horn, cattle horn, and pig hoof.

[0017] Beneficial effects: The present invention screened out a characteristic peptide of antelope horn through a large number of experimental screenings. This characteristic peptide of antelope horn has high specificity and can be used to identify medicines containing antelope horn medicinal materials.

[0018] The antelope horn characteristic peptide and the detection method thereof provided by the present invention screen out the best chromatographic conditions and mass spectrometry conditions through a large number of experiments. The whole method is simple to operate, accurate in judgment, and can accurately distinguish the antelope horn characteristic peptide. The antelope horn characteristic peptide and the detection method thereof provided by the present invention are conducive to the quality control of antelope horn medicines, and are of great significance for ensuring the quality of medicines containing antelope horns. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the XIC diagram of the characteristic peptide of antelope horn. DETAILED DESCRIPTION

[0020] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0021] Example 1

[0022] A characteristic peptide segment of antelope horn, the characteristic peptide sequence is shown in Sequence Table 1:

[0023] Characteristic peptide: Ile-Ser-Pro-Gly-Tyr-Ser-Val-Thr-Arg.

[0024] Example 2 Detection of characteristic polypeptides in antelope horns

[0025] (1) Take 10 mg of antelope horn, yellow cattle horn, and pig hoof nail samples, add 1 ml of protein lysis buffer [4% SDS, 20 mM DTT, 50 mM Tris-HCl (pH = 8.8)] respectively, homogenize 3 times, each time for 60 seconds, centrifuge at 12000 rpm for 20 minutes, and determine the protein content by BCA. Take 1000 μg of protein, add 25 μl of 0.2 M IAA solution, keep it away from light for 30 minutes, and add 5.5V 样品 80% cold acetone solution, mix thoroughly and freeze at -20℃ for precipitation overnight.

[0026] (2) Take the acetone precipitate samples from step (1) respectively, centrifuge at 12000rpm for 20min, discard the supernatant, wash the precipitate once with 80% cold acetone, evaporate the precipitate, add 200μl 8M urea solution to dissolve it, shake it thoroughly, and after the precipitate is completely dissolved, add 1400μl Tris-HCl buffer to reduce the urea concentration to below 1M, add 40μl of 0.1μg / μl trypsin to each horn sample, incubate in a constant temperature water bath at 37°C for 12h, and obtain the enzymatic hydrolyzate of each horn sample.

[0027] (3) Prepare 1 ml of a 200 ng / ml reference solution of the purified characteristic peptide described in Example 1, add 5 μl of 0.2 M IAA solution, and place in a dark place for 30 min.

[0028] (4) The enzymatic hydrolysates of each horn species in step (2) and the characteristic peptide reference solution treated with IAA were detected by liquid chromatography-mass spectrometry, and the liquid phase conditions were as follows: the chromatographic column was a 1.8 μm Waters T3 column with a specification of 2.1 μm×100 mm, the sample volume was 10 μl, the flow rate was 0.3 ml / min, the mobile phase A was 0.1% formic acid, the mobile phase B was acetonitrile, 0-5 min, 5-20% B linear gradient elution, 5-8 min, 20-95% B linear gradient elution, 8-9 min, 95% B isocratic elution; a triple quadrupole mass spectrometer was used, and the mass spectrometry parameters were as follows: ion source temperature 500°C; ionization voltage 5500 V; desolvation temperature 500°C; ion source gas 1, 60 psi; ion source gas 2, 60 psi. The ion pairs in the MRM mode were: m / z 490.3 double charge → 779.4, DP=40, CE=20.

[0029] Results Figure 1 (In the figure, (A) is the reference substance; (B) is antelope horn; (C) is cattle horn; (B) is pig hoof nail). An ion peak consistent with the characteristic peptide reference solution can be detected in the antelope horn sample, but not in other samples. This indicates that the present invention can specifically detect the characteristic peptide components in antelope horn, thereby distinguishing it from other keratinous animal drugs.

[0030] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A characteristic peptide segment of antelope horn, characterized in that: The characteristic peptide segment is: Ile-Ser-Pro-Gly-Tyr-Ser-Val-Thr-Arg.

2. A method for detecting characteristic peptides of antelope horns, characterized in that: The following steps are involved: (1) preparing a reference solution using the characteristic antelope horn peptide described in claim 1; (2) After the sample to be tested is digested with trypsin, the enzymatic hydrolyzate and the antelope horn characteristic peptide reference solution of step (1) are respectively injected into a liquid spectrometer, and the antelope horn characteristic peptide is used as a reference, and the detection is performed in a multiple reaction monitoring mode; if an antelope horn characteristic peptide ion pair is detected, and the retention time of the ion is consistent with that of the antelope horn characteristic peptide reference, and its daughter ion is consistent with the daughter ion of the antelope horn characteristic peptide reference, then the sample to be tested contains antelope horn components.

3. The method for detecting characteristic peptides of antelope horns according to claim 2, characterized in that: The enzyme digestion method is performed as follows: 10 mg of the horn animal drug sample to be tested is taken, 1 ml of protein lysis solution is added, homogenized 3 times, centrifuged, the supernatant is taken, 1000 μg of protein is taken, 0.2 M IAA solution is added, placed away from light, cold acetone solution is added, after being fully mixed, refrigerated, and precipitated overnight; After centrifugation of the acetone precipitate sample, discard the supernatant, wash the precipitate once with cold acetone, evaporate the precipitate, add urea solution to dissolve it, shake it thoroughly, add Tris-HCl buffer after the precipitate is completely dissolved, add trypsin to each horn sample respectively, and incubate in a constant temperature water bath to obtain the product.

4. The method for detecting characteristic peptides of antelope horns according to claim 3, characterized in that: The enzyme cleavage method is as follows: take 10 mg of the horn animal drug sample to be tested, add 1 ml of protein lysis solution, add grinding beads, homogenize 3 times, each time for 60 seconds, centrifuge at 12000 rpm for 20 minutes, determine the protein content by BCA, take 1000 μg of protein, add 25 μl of 0.2MIAA solution, place in dark for 30 minutes, add 5.5V 样品 80% cold acetone solution, after fully mixing, freeze and precipitate at -20°C overnight to obtain an acetone precipitation sample; the pH value of the protein lysis solution is 8.8, which includes 4% sodium dodecyl sulfate, 20mM dithiothreitol, and 50mM Tris-HCl; Take the acetone precipitate sample, centrifuge it at 12000rpm for 20min, discard the supernatant, wash the precipitate once with 80% cold acetone, evaporate the precipitate, add 200μl 8M urea solution to dissolve it, shake it thoroughly, add 1400μl Tris-HCl buffer after the precipitate is completely dissolved to reduce the urea concentration to below 1M, add 40μl of 0.1μg / μl trypsin to the sample, and incubate it in a constant temperature water bath at 37℃ for 12h.

5. The method for detecting characteristic peptides of antelope horns according to claim 2, characterized in that: The liquid phase conditions for LC-MS detection are as follows: the chromatographic column is a 1.8μm Waters T3 column with a specification of 2.1μm×100mm, the sample volume is 10μl, the flow rate is 0.3ml / min, the mobile phase A is 0.1% formic acid, the mobile phase B is acetonitrile, 0-5min, 5-20% B linear gradient elution, 5-8min, 20-95% B linear gradient elution, 8-9min, 95% B isocratic elution; a triple quadrupole mass spectrometer is used, and the mass spectrometer parameters are: ion source temperature 500℃; ionization voltage 5500V; desolvation temperature 500℃; ion source gas 1, 60psi; ion source gas 2, 60psi; the ion pairs of the multiple reaction monitoring mode MRM are: m / z 490.3 doubly charged→779.4, DP=40, CE=20.

6. The method for detecting characteristic peptides of antelope horns according to claim 2, characterized in that: Horny animal medicines include antelope horns, cattle horns, and pig hoofs.

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