Characteristic peptide fragment for identifying antelope horn and detection method thereof

Abstract

The invention discloses a characteristic peptide fragment for identifying antelope's horn and a detection method thereof, and the method comprises the following steps: screening out two sequences through a large number of experiments, namely Val-Val-Ser-Thr-His-Glu-Gln-Ile-Val-Arg; the antelope horn characteristic peptide fragments of Lu-Ala-Ser-Tyr-Leu-Glu-Lys have very 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 fragment, 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 fragment can be accurately distinguished. The antelope horn characteristic peptide fragment 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 is the horn of Saiga tatarica Linnaeus, a bovine animal. It is one of the precious and rare Chinese medicines in my country. As early as the "Shennong Bencao Jing", there was a record of antelope horn being used as medicine. It has a long history of medicinal use and plays an indispensable role in the clinical practice of traditional Chinese medicine and the Chinese medicine industry. It has made important contributions to the survival and reproduction of the Chinese nation. It tastes salty and is cold in nature. It enters the liver and heart meridians. It has the effects of calming the liver and calming the wind, clearing the liver and improving eyesight, dispersing blood and detoxifying. It is mainly used to treat diseases such as liver wind movement, epilepsy and convulsions, high fever convulsions, red eye cataracts, fever, carbuncle, sore and poison. Modern pharmacological studies have shown that antelope horn has pharmacological activities such as antipyretic, anticonvulsant, analgesic, sedative, and hypotensive. Chinese patent medicines containing antelope horn are mainly used to treat stroke, headache and dizziness, palpitations, exogenous wind-heat, epilepsy and convulsions, red eye cataracts, high fever and other diseases, and have clear clinical application value. At present, the quality standard of antelope horn is not perfect. There is a lack of specific identification and content determination methods for antelope horn medicinal materials and prescription preparations containing antelope horn. There is a phenomenon of mixing with other horns or hoofs in the medicinal material market. Modern research shows that antelope horn contains rich protein and peptide components, which are not only the structural substances of antelope horn, but also important components of its functional substances. Therefore, the present invention uses antelope horn characteristic peptides to detect antelope horn medicinal materials, and can be used to distinguish other mixed and counterfeit products. This method provides a reference and basis for the improvement of antelope horn quality standards. Summary of the invention

[0003] The main purpose of the present invention is to provide a method for detecting characteristic peptides of antelope horns in view of the above-mentioned problems and shortcomings. The characteristic peptides of antelope horns provided by the present invention have strong specificity. The provided method is simple to operate and has high sensitivity. It can accurately identify whether the medicine contains antelope horn ingredients, and provides 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] Two characteristic peptides of antelope horns, the sequences of which are shown in Sequence Table 1:

[0006] Characteristic peptide 1: Val-Val-Ser-Thr-His-Glu-Gln-Ile-Val-Arg;

[0007] Characteristic peptide 2: Leu-Ala-Ser-Tyr-Leu-Glu-Lys.

[0008] The method for detecting characteristic peptides of antelope horns comprises the following steps:

[0009] (1) preparing a reference solution with antelope horn characteristic peptides 1 and 2;

[0010] (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 the above-mentioned 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 the daughter ion of the reference substance, then the sample to be tested contains antelope horn components.

[0011] As a preferred embodiment, the above-mentioned method for detecting characteristic peptides of antelope horns, the enzymatic cleavage method is carried out as follows: take 10g of the horn animal drug sample to be detected, add 100ml of water to extract, centrifuge, take the supernatant, determine the protein content of the extract, add trypsin at a protein amount of 1:50, enzymatically hydrolyze, add 0.2M IAA solution, place in dark for 30 minutes, desalt, evaporate to dryness, and obtain.

[0012] As a preferred embodiment, the above-mentioned method for detecting characteristic peptides of antelope horns, the enzymatic cleavage method is carried out as follows: take 10g of the horn animal drug sample to be detected, add 100ml of water and reflux extraction for 30min, extract twice, combine the filtrate, centrifuge at 12000rpm for 20min, take the supernatant, determine the protein content by BCA, take 400μg of protein, freeze-dry, and obtain lyophilized powder of the extract; the lyophilized powder is re-dissolved with 100μl Tris-HCl buffer, and after re-dissolution, 100μl of 0.1μg / μl trypsin is added, after ultrasonic enzymatic hydrolysis for 1 hour, 50μl of 0.2M IAA solution is added, and the mixture is placed away from light for 30min. After treatment with a C18 desalting column, it is evaporated to obtain the extract.

[0013] As a preferred embodiment, the detection method of the characteristic peptide fragments 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, 5-10% B linear gradient elution, 5-7min, 10-15% B linear gradient elution, 7-8min, 15-95% 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.

[0014] The ion pairs in MRM mode are characteristic peptide 1: m / z 584.3 doubly charged→969.5, DP=130, CE=40; characteristic peptide 2: m / z 412.2 doubly charged→639.3, DP=40, CE=20.

[0015] As a preferred embodiment, the above-mentioned method for detecting characteristic peptide segments of antelope horns is characterized in that the horn animal drugs include antelope horns, cattle horns, and pig hoofs.

[0016] Beneficial effects: The present invention screened out two characteristic peptides of antelope horn through a large number of experimental screenings. These two characteristic peptides of antelope horn have high specificity and can be used to identify medicines containing antelope horn medicinal materials.

[0017] The present invention hydrolyzes antelope horns, and screens out characteristic peptides that can specifically identify antelope horns through chromatographic classification and mass spectrometry detection. In addition, the present invention screens out the best chromatographic conditions and mass spectrometry conditions for identifying characteristic peptides of antelope horns through a large number of experiments. The entire method is simple to operate, accurate in judgment, and can accurately distinguish characteristic peptides of antelope horns. The characteristic peptides of antelope horns 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

[0018] Figure 1 This is the XIC diagram of the characteristic peptide 1 of antelope horn;

[0019] Figure 2 This is the XIC diagram of the characteristic peptide 2 from 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] Two characteristic peptides of antelope horns, the sequences of which are shown in Sequence Table 1:

[0023] Characteristic peptide 1: Val-Val-Ser-Thr-His-Glu-Gln-Ile-Val-Arg;

[0024] Characteristic peptide 2: Leu-Ala-Ser-Tyr-Leu-Glu-Lys.

[0025] Example 2 Detection of characteristic peptides in antelope horns

[0026] (1) Take 10 mg of each sample of antelope horn, yellow cattle horn, and pig hoof nail, add 100 ml of water respectively, reflux and extract for 30 minutes, extract twice, combine the filtrate, centrifuge at 12000 rpm for 20 minutes, take the supernatant, determine the protein content by BCA, take 400 μg of protein, freeze-dry, and obtain the extract lyophilized powder; the lyophilized powder is reconstituted with 100 μl of Tris-HCl buffer, and after reconstitution, 100 μl of 0.1 μg / μl trypsin is added. After ultrasonic enzymatic hydrolysis for 1 hour, 50 μl of 0.2 M IAA solution is added, and the mixture is placed away from light for 30 minutes. After treatment with a C18 desalting column, it is evaporated to obtain the enzymatic hydrolyzate of each horn sample.

[0027] (2) Prepare 1 ml of a 200 ng / ml reference solution of the purified characteristic peptide described in Example 1, add 5 μl of a 0.2 M IAA solution, and place in a dark place for 30 min.

[0028] (3) The enzymatic hydrolysates of each horn species in step (1) and the characteristic peptide reference solution treated with IAA in step (2) 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, 5-10% B linear gradient elution, 5-7 min, 10-15% B linear gradient elution, 7-8 min, 15-95% 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 MRM mode are characteristic peptide 1: m / z584.3 doubly charged→969.5, DP=130, CE=40; characteristic peptide 2: m / z412.2 doubly charged→639.3, DP=40, CE=20.

[0029] Results Figure 1 (In the figure, (A) is the reference substance of characteristic peptide 1; (B) is antelope horn; (C) is cattle horn; (D) is pig hoof nail); Figure 2 (In the figure, (A) is the characteristic peptide 2 reference substance; (B) is the antelope horn; (C) is the cattle horn; and (D) is the pig hoof nail). Ion peaks consistent with the characteristic peptide 1 and characteristic peptide 2 reference substance solutions 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 the 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 for identifying antelope horns, characterized in that: The characteristic peptide sequence is: Characteristic peptide 1: Val-Val-Ser-Thr-His-Glu-Gln-Ile-Val-Arg; Characteristic peptide 2: Leu-Ala-Ser-Tyr-Leu-Glu-Lys.

2. A detection method for identifying characteristic peptide segments 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 hydrolysate 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 the above-mentioned 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 reference, then the sample to be tested contains antelope horn components.

3. The method for detecting characteristic peptide segments of antelope horns according to claim 2, characterized in that: The enzymatic cleavage method is carried out as follows: take 10g of the horn animal drug sample to be tested, add 100ml of water to extract, centrifuge, take the supernatant, determine the protein content of the extract, add trypsin at a protein amount of 1:50, enzymatically hydrolyze, add 0.2M IAA solution, place in the dark for 30 minutes, desalt, evaporate to dryness, and obtain.

4. The method for detecting characteristic peptide segments of antelope horns according to claim 3, characterized in that: The enzymatic cleavage method is carried out as follows: take 10g of the horn animal drug sample to be tested, add 100ml of water and reflux extract for 30min, extract twice, combine the filtrate, centrifuge at 12000rpm for 20min, take the supernatant, determine the protein content by BCA, take 400μg of protein, freeze-dry, and obtain the extract lyophilized powder; the lyophilized powder is re-dissolved with 100μl Tris-HCl buffer, and after re-dissolution, 100μl of 0.1μg / μl trypsin is added, after ultrasonic enzymatic hydrolysis for 1 hour, 50μl 0.2M IAA solution is added, and it is placed away from light for 30min. After being treated with a C18 desalting column, it is evaporated to obtain the extract.

5. The method for detecting characteristic peptide segments 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, 5-10% B linear gradient elution, 5-7min, 10-15% B linear gradient elution, 7-8min, 15-95% 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°C; ionization voltage 5500V; desolvation temperature 500°C; ion source gas 1, 60psi; ion source gas 2, 60psi. The ion pairs in MRM mode are characteristic peptide 1: m / z 584.3 doubly charged→969.5, DP=130, CE=40; characteristic peptide 2: m / z 412.2 doubly charged→639.3, DP=40, CE=20.

6. The method for detecting characteristic peptide segments 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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