Characteristic peptide fragment for identifying antelope horn and detection method thereof

Abstract

The invention discloses a characteristic peptide fragment for identifying antelope's horns and a detection method of the characteristic peptide fragment. The sequence of the characteristic peptide fragment is Ala-Gln-Tyr-Asp-Asp-Ile-Ala-Ser-Ar, and the sequence of the characteristic peptide fragment is Leu-Val-Val-Gln-Ile-Asp-Asn-Ala-Lys. The characteristic peptide fragment is used for identifying the antelope's horns. Different types of peptides are obtained through a large number of experimental screening and enzymolysis of antelope horn protein, then two characteristic peptide fragments special for identifying antelope horns are screened out, and the two characteristic peptide fragments of the antelope horns have high specificity and can be used for identifying drugs containing antelope horn medicinal materials and distinguishing the drugs from other cornu bubali and pig nails. According to the detection method for identifying the characteristic peptide fragment of the antelope horn, the optimal chromatographic condition and mass spectrum condition are screened out through a large number of experiments, the method is high in sensitivity and accuracy and good in reproducibility and stability, and the detection method is beneficial to quality control of an antelope horn medicine and has good application prospects. And the method has important significance for ensuring the quality of the antelope horn-containing medicine.

Description

Technical Field

[0001] The invention relates to a detection and identification method for animal drugs, and in particular to a characteristic peptide segment for identifying antelope horns 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 horn shows that its main components are proteins, peptides, amino acids, sterols, nucleosides, and inorganic elements.

[0003] The current quality standards for antelope horns are not perfect. Antelope horn medicinal materials, concentrated powders, and prepared preparations containing antelope horns lack specific identification and content measurement methods. Other horns or hooves are also mixed and sold in the medicinal materials market.

[0004] According to the current status of the existing technology, the present invention screens the characteristic peptides of antelope horn hydrolysate and finds characteristic peptides that can be used to detect antelope horn components. This method provides a reference and basis for the quality research of antelope horn, which is conducive to further improvement of the quality standards of antelope horn and its products. Summary of the invention

[0005] The main purpose of the present invention is to provide a characteristic peptide segment and a detection method thereof for identifying antelope horns in view of the above-mentioned problems and shortcomings.

[0006] Technical solution: In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] A characteristic peptide segment for identifying antelope horns, the sequence of the characteristic peptide segment is shown in Sequence Table 1:

[0008] Peptide I: Ala-Gln-Tyr-Asp-Asp-Ile-Ala-Ser-Arg

[0009] Peptide II: Leu-Val-Val-Gln-Ile-Asp-Asn-Ala-Lys.

[0010] A detection method for identifying characteristic peptide segments of antelope horns, comprising the following steps:

[0011] (1) preparing the characteristic peptide segments for identifying antelope horns according to claim 1 into reference solution;

[0012] (2) After the sample to be tested is double-enzymed with LysC and trypsin, the enzymatic hydrolysate and the characteristic peptide reference solution for identifying antelope horn in step (1) are respectively injected into a liquid spectrometer, and the characteristic peptide for identifying antelope horn is used as a control, and the detection is carried out 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 characteristic peptide reference for identifying antelope horn, and its daughter ion is consistent with the daughter ion of the reference, then the sample to be tested contains antelope horn components.

[0013] As a preferred embodiment, the above-mentioned detection method for identifying the characteristic peptide segments of antelope horns, the enzymatic cleavage method is carried out as follows: take 10 mg of the horn animal drug sample to be detected, add 1 ml of phosphate buffer, homogenize 3 times, centrifuge, first add LysC protease, incubate in a constant temperature water bath at 37°C, centrifuge to take the supernatant, take 1000 μg of protein, then add trypsin, incubate in a constant temperature water bath at 37°C, and obtain.

[0014] As a preferred embodiment, the above-mentioned detection method for identifying the characteristic peptide segments of antelope horns, the enzymatic cleavage method is carried out as follows: take 10 mg of the horn animal drug sample to be detected, add 1 ml of phosphate buffer, add grinding beads, homogenize 3 times, each time for 60 seconds, add 40 μl of 0.1 μg / μl LysC protease, incubate in a constant temperature water bath at 37°C for 6 hours, centrifuge at 12000 rpm for 20 minutes, determine the protein content by BCA, take 1000 μg of protein, add 100 μl of 0.1 μg / μl trypsin, and incubate in a constant temperature water bath at 37°C for 4 hours.

[0015] As a preferred embodiment, the above-mentioned detection method for identifying the characteristic peptide segments of antelope horns, the liquid phase conditions for liquid chromatography-mass spectrometry detection are: the chromatographic column is a 1.8μm Waters T3 column, the specification is 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, 0-7min, 10-30% B linear gradient elution, 7-8min, 30-90% B linear gradient elution, 8-9min, 90% 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; the selected multiple reaction monitoring mode conditions are: the ion pair of the MRM mode of peptide I is: m / z 519.7 doubly charged→676.3, DP=40, CE=20; the ion pair of MRM mode of peptide II is: m / z 500.3 doubly charged→787.4, DP=40, CE=20.

[0016] As a preferred embodiment, in the above-mentioned detection method for identifying characteristic peptide segments of antelope horns, horn animal drugs include antelope horns, cattle horns, pig hoofs, etc.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0018] The present invention obtains different types of peptides by enzymatically hydrolyzing antelope keratin through a large number of experiments and then screens out two characteristic peptide segments specifically used for identifying antelope horns. The two characteristic peptide segments of antelope horns have high specificity and can be used to identify medicines containing antelope horn medicinal materials and distinguish them from other cattle horns and pig hoofs.

[0019] The detection method for identifying characteristic peptide segments of antelope horn provided by the present invention screens out the best chromatographic conditions and mass spectrometry conditions through a large number of experiments. The method has high sensitivity and accuracy, good reproducibility and stability. The detection method is conducive to the quality control of antelope horn medicines and is of great significance for ensuring the quality of medicines containing antelope horn. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The XIC diagram of the characteristic peptide I and the sample used to identify antelope horn.

[0021] Figure 2 The XIC diagram of the characteristic peptide II and samples used to identify antelope horns. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1

[0024] A characteristic peptide segment for identifying antelope horns, the sequence of the characteristic peptide segment is shown in Sequence Table 1:

[0025] Peptide I: Ala-Gln-Tyr-Asp-Asp-Ile-Ala-Ser-Arg

[0026] Peptide II: Leu-Val-Val-Gln-Ile-Asp-Asn-Ala-Lys.

[0027] Example 2 Detection of characteristic polypeptides in antelope horns

[0028] (1) Take 10 mg of antelope horn, ox horn, and pig hoof nail samples, add 1 ml of phosphate buffer, add grinding beads, homogenize 3 times, each time for 60 seconds, add 40 μl of 0.1 μg / μl LysC protease, incubate in a constant temperature water bath at 37°C for 6 hours, centrifuge at 12000 rpm for 20 minutes, determine the protein content by BCA, take 1000 μg of protein, add 100 μl of 0.1 μg / μl trypsin, incubate in a constant temperature water bath at 37°C for 4 hours, and obtain the enzymatic hydrolysate of each horn sample.

[0029] (2) Prepare 1 ml of 200 ng / ml concentration of the pure characteristic peptide described in Example 1 as a reference solution, add 5 μl of 0.2 M IAA solution, and place in the dark for 30 min.

[0030] (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, 0-7 min, 10-30% B linear gradient elution, 7-8 min, 30-90% B linear gradient elution, 8-9 min, 90% 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 selected multiple reaction monitoring mode conditions were: the ion pairs of MRM mode for peptide I were: m / z 519.7 doubly charged→676.3, DP=40, CE=20; the ion pairs of MRM mode for peptide II were: m / z 500.3 doubly charged→787.4, DP=40, CE=20.

[0031] Test results: Figure 1 and Figure 2 (In the figure, A is a peptide reference; B is an antelope horn; C is a yellow cattle horn; and D is a pig hoof nail). In the antelope horn sample, ion peaks m / z519.7 double charged→676.3 and m / z500.3 double charged→787.4, which are consistent with the characteristic peptide reference solution, can be detected, 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.

[0032] 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 for identifying antelope horns, characterized in that: The characteristic peptide segment is: Peptide I: Ala-Gln-Tyr-Asp-Asp-Ile-Ala-Ser-Arg Peptide II: Leu-Val-Val-Gln-Ile-Asp-Asn-Ala-Lys.

2. A detection method for identifying characteristic peptide segments of antelope horns, characterized in that: The following steps are involved: (1) preparing the characteristic peptide segments for identifying antelope horns as claimed in claim 1 into reference solution; (2) After the sample to be tested is double-enzymed with LysC and trypsin, the enzymatic hydrolysate and the characteristic peptide reference solution for identifying antelope horn in step (1) are respectively injected into a liquid spectrometer, and the characteristic peptide for identifying antelope horn is used as a control, and the detection is carried out 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 characteristic peptide reference for identifying antelope horn, 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 enzyme cleavage method is carried out as follows: take 10 mg of the horn animal drug sample to be tested, add 1 ml of phosphate buffer, homogenize 3 times, centrifuge, first add LysC protease, incubate in a constant temperature water bath at 37°C, centrifuge to take the supernatant, take 1000 μg of protein, then add trypsin, and incubate in a constant temperature water bath at 37°C to obtain the product.

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 10 mg of the horn animal drug sample to be tested, add 1 ml of phosphate buffer, add grinding beads, homogenize 3 times, each time for 60 seconds, add 40 μl of 0.1 μg / μl LysC protease, incubate in a constant temperature water bath at 37°C for 6 hours, centrifuge at 12000 rpm for 20 minutes, determine the protein content by BCA, take 1000 μg of protein, add 100 μl of 0.1 μg / μl trypsin, and incubate in a constant temperature water bath at 37°C for 4 hours.

5. The method for detecting characteristic peptide segments for identifying antelope horns according to claim 2, characterized in that: The liquid phase conditions for LC-MS detection 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, 0-7 min, 10-30% B linear gradient elution, 7-8 min, 30-90% B linear gradient elution, 8-9 min, 90% B isocratic elution; a triple quadrupole mass spectrometer was used, and the mass spectrometer 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 selected multiple reaction monitoring mode conditions were as follows: the ion pair of the MRM mode of peptide I was: m / z 519.7 doubly charged → 676.3, DP=40, CE=20; the ion pair of the MRM mode of peptide II was: m / z 500.3 double charge → 787.4, 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, pig hoofs, and buffalo horns.

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