CN115160405B - Cornu Saigae Tataricae characteristic peptide fragment and detection method thereof - Google Patents
Cornu Saigae Tataricae characteristic peptide fragment and detection method thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a antelope horn characteristic peptide fragment and a detection method thereof, and the antelope horn characteristic peptide fragment is screened out by a large number of experiments, has high specificity and can be used for identifying medicines containing antelope horn medicinal materials. According to the method for detecting the characteristic peptide of the antelope horn, provided by the invention, the optimal chromatographic conditions and mass spectrum conditions are screened out through a large number of experiments, the whole method is simple to operate, the judgment is accurate, and the characteristic peptide of the antelope horn can be accurately distinguished. The antelope horn characteristic peptide and the detection method thereof provided by the invention are beneficial to quality control of antelope horn medicines and have important significance for ensuring the quality of medicines containing antelope horn.
Description
Technical Field
The invention relates to a detection and identification method of animal medicines, in particular to a characteristic peptide segment of antelope horn and a detection method thereof.
Background
Cornu Saigae Tataricae SAIGAE TATARICAE Cornu is cornu Saigae Tataricae SAIGA TATARICA Linnaeus of bovine, and is originally carried in Shennong Ben Cao Jing, so-called sheep, commonly called cornu Saigae Tataricae, with the effects of: cold nature, salty taste, liver-pacifying, wind-extinguishing, heat-clearing, convulsion-relieving, detoxifying; is generally used for treating various febrile diseases, such as high fever headache, coma, delirium mania, apoplexy, convulsion and macula, etc., and is an important variety in the clinical and pharmaceutical industries of traditional Chinese medicine. Modern pharmacological researches have shown that the antelope horn has good effects in tranquilizing, hypnotizing, anticonvulsant, analgesic, antiepileptic, anti-inflammatory, antipyretic, antipathogenic microorganism, antithrombotic, vascular permeability changing, antihypertensive, antitussive, expectorant and immunity enhancing. The clinical and pharmaceutical industries of traditional Chinese medicine apply the antelope horn to Chinese patent medicines: the compound antelope horn antihypertensive tablet, the antelope lung-heat clearing pill, the antelope anti-inflammatory pill and the like have better curative effect in clinical application. Basic researches on antelope horn substances show that proteins, peptides, amino acids, sterols, nucleosides, inorganic elements and the like are taken as main components. The quality standard of the antelope horn is imperfect, and the antelope horn medicinal material, the concentrated powder and the formulated preparation containing the antelope horn lack a specific identification and content determination method, and the medicinal material market has the phenomenon of being sold in other horns or ungulates in a mixed way. The research shows that the DNA bar code technology based on the COI sequence can be used for identifying the antelope horn medicinal material mixed and counterfeited product, but the high-temperature extraction process of the traditional Chinese medicine can influence the integrity of DNA, so that the identification accuracy of the DNA bar code is further influenced, compared with the DNA molecule, the primary sequence information of protein and peptide components is better preserved under the high-temperature extraction condition, and protein components such as the antelope horn, buffalo horn, goat horn and the like are important substance bases of the animal medicines such as the Type I Keratin (Keratin, type I), the Type II Keratin (Type I Collagen) and the like. The method detects the antelope horn components by detecting 1 antelope horn characteristic peptide, provides reference and basis for the quality research of the antelope horn, and is favorable for further perfecting the quality standard of the antelope horn and the product thereof.
Disclosure of Invention
The invention mainly aims at solving the problems and the defects and provides a characteristic peptide of the antelope horn and a method for detecting the characteristic peptide of the antelope horn in a horn-like animal medicine. The antelope horn characteristic peptide provided by the invention has strong specificity. The provided method is simple to operate and high in sensitivity, can accurately identify whether the horn-like animal medicine contains the antelope horn component, and provides a scientific method for ensuring the quality of the antelope horn and the product thereof.
The technical scheme is as follows: in order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the characteristic peptide fragment of the antelope horn has a characteristic peptide sequence as shown in a sequence table 1:
characteristic peptide: leu-Lys-Gln-Glu-Val-Asn-Cys-Ala-Tyr-Val-Arg
A detection method of antelope horn characteristic peptide comprises the following steps:
(1) Preparing antelope horn characteristic peptide into reference substance solution;
(2) After enzyme digestion is carried out on a horn-like animal medicine sample to be detected by trypsin, the enzymolysis liquid and the antelope horn characteristic peptide mixed reference substance solution in the step (1) are injected into a liquid-mass spectrometer, the antelope horn characteristic peptide is used as a reference, a multi-reaction monitoring mode is adopted, and the selection conditions are as follows: m/z 690.4 (double charge) → 782.3 (dp=85.08, ce= 40.88), m/z 690.4 (double charge) → 881.2 (dp=101.67, ce=39.06), and detection is performed; if the ion pair is detected, the retention time of the ions is consistent with that of the antelope horn characteristic peptide reference substance, and the sub-ions of the ion pair are consistent with that of the reference substance, the sample to be detected contains antelope horn components.
As a preferred scheme, the method for detecting the characteristic peptide of the antelope horn is carried out as follows: taking 20mg of a horn-like animal medicine sample to be detected, adding 1ml of protein lysate, homogenizing for 3 times, centrifuging, taking supernatant, adding 0.2M IAA solution, placing in a dark place, adding cold acetone solution, fully and uniformly mixing, refrigerating, and precipitating overnight;
centrifuging an acetone precipitate sample, discarding supernatant, washing the precipitate once with cold acetone, volatilizing the precipitate, adding urea solution for re-dissolution, sufficiently shaking, adding Tris-HCl buffer solution after the precipitate is completely dissolved, respectively adding trypsin into each corner sample, and incubating in a constant-temperature water bath.
As a preferred scheme, the method for detecting the characteristic peptide of the antelope horn is carried out as follows: taking 20mg of a sample of the horn-like animal medicine to be detected, and adding 1ml of protein lysate (comprising 4% Sodium Dodecyl Sulfate (SDS), 20mM Dithiothreitol (DTT), 50mM Tris-HCl, pH=8.8); adding grinding beads, homogenizing for 3 times, centrifuging at 12000rpm for 20min each time for 60s, measuring protein content by BCA, taking 1000 μg protein, adding 25 μl 0.2M IAA solution, standing in dark for 30min, adding 5.5V Sample of % cold acetone solution, mixing completely, and standing at-20deg.C for freezing and precipitating overnight;
Taking an acetone precipitate sample, centrifuging at 12000rpm for 20min, discarding the supernatant, washing the precipitate twice with 80% cold acetone, volatilizing the precipitate, adding 200 μl of 8M urea solution for redissolution, sufficiently shaking, adding 1400 μl of Tris-HCl buffer solution after the precipitate is completely dissolved, reducing the urea concentration to below 1M, respectively adding 0.1 μg/μl of trypsin 40 μl into each corner sample, and incubating for 12h in a constant-temperature water bath at 37 ℃ to obtain the final product.
As a preferred scheme, the method for detecting the characteristic peptide of the antelope horn comprises the following steps of: the chromatographic column is a 1.7 mu m Waters C 18 column, the specification is 2.1 mu m multiplied by 50mm, the loading amount is 10 mu l, the flow rate is 0.3ml/min, the mobile phase A is 0.1% formic acid, the mobile phase B is 0.1% acetonitrile formate, the linear gradient elution is carried out for 0 to 3.5min,5 to 20% B, the linear gradient elution is carried out for 3.5 to 4min,20 to 5% B, the isocratic elution is carried out for 4 to 6min and 5% B; by adopting triple quadrupole mass spectrometry, electrospray positive ion mode ESI+ is adopted, and mass spectrometry parameters are as follows: the temperature of the ion source is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60psi; ion source gas 2, 60psi.
The ion pairs for MRM mode are: m/z 690.4 double charge→ 782.3 (dp=85.08, ce= 40.88), m/z 690.4 double charge→ 881.2 (dp=101.67, ce=39.06).
In a preferred embodiment, the method for detecting the characteristic peptide of cornu Saigae Tataricae is characterized in that the horn-like animal medicine comprises cornu Saigae Tataricae, cornu Naemorhedi, cornu Bubali, and ungula Sus domestica.
The beneficial effects are that: according to the invention, 1 antelope horn characteristic peptide is screened out through a large number of experimental screening, and the 1 antelope horn characteristic peptide has high specificity and can be used for identifying medicines containing antelope horn medicinal materials.
According to the antelope horn characteristic peptide and the detection method thereof, the optimal chromatographic conditions and mass spectrum conditions are screened out through a large number of experiments. The whole method is simple to operate, accurate in judgment and capable of accurately distinguishing the characteristic peptide of the antelope horn. The antelope horn characteristic peptide and the detection method thereof provided by the invention are beneficial to quality control of antelope horn medicines and have important significance for ensuring the quality of medicines containing antelope horn.
Drawings
FIG. 1 is a diagram of the XIC of a characteristic peptide of cornu Saigae Tataricae;
Fig. 2 is an XIC view of the antelope horn peptide of character.
Detailed Description
The present application is further illustrated below with reference to specific examples, which are to be construed as merely illustrative of the application and not limiting of its scope, as various equivalent modifications to the application will fall within the scope of the application as defined in the appended claims after reading the application.
Example 1
The characteristic peptide fragment of the antelope horn has a characteristic peptide sequence as shown in a sequence table 1:
characteristic peptide: leu-Lys-Gln-Glu-Val-Asn-Cys-Ala-Tyr-Val-Arg.
EXAMPLE 2 detection of characteristic Polypeptides in various keratomes
(1) Taking 20mg of antelope horn, buffalo horn, yak horn, yellow ox horn and pig's feet, respectively adding 1ml of protein lysate [4%SDS,20mM DTT,50mM Tris-HCl (pH=8.8) ], homogenizing for 3 times, centrifuging at 12000rpm for 20min each time, measuring protein content by BCA, taking 1000 mu g of protein, adding 25 mu l of 0.2M IAA solution, placing for 30min in a dark place, adding 5.5V Sample of % cold acetone solution, fully mixing, and standing at-20 ℃ for freezing and precipitating overnight.
(2) Respectively taking acetone precipitation samples in the step (1), centrifuging at 12000rpm for 20min, discarding the supernatant, washing the precipitate once with 80% cold acetone, volatilizing the precipitate, adding 200 μl of 8M urea solution for re-dissolution, sufficiently shaking, adding 1400 μl of Tris-HCl buffer solution after the precipitate is completely dissolved to reduce the urea concentration to below 1M, respectively adding 0.1 μg/μl of trypsin 40 μl into each corner sample, and incubating for 12h in a constant-temperature water bath at 37 ℃ to obtain each corner enzymatic hydrolysate.
(3) The purified characteristic peptide of example 1 was prepared as a control solution at a concentration of 200ng/ml in 1ml, and 5. Mu.l of 0.2M IAA solution was added thereto and left in a dark place for 30min.
(4) Detecting the corner enzymolysis liquid in the step (2) and the characteristic peptide reference substance solution treated by IAA by adopting a liquid chromatography-mass spectrometer, wherein the liquid phase condition is as follows: the chromatographic column is a 1.7 μm Waters C 18 column (2.1 μm. Times.50 mm), the loading is 10. Mu.l, the flow rate is 0.3ml/min, the mobile phase A is 0.1% formic acid, the mobile phase B is 0.1% acetonitrile formate, the linear gradient elution is carried out for 0 to 3.5min,5 to 20% B, the linear gradient elution is carried out for 3.5 to 4min,20 to 5% B, the isocratic elution is carried out for 4 to 6min and 5% B. Triple quadrupole mass spectrometry is adopted, and mass spectrometry parameters are as follows: the temperature of the ion source is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60psi; ion source gas 2, 60psi. The ion pairs for MRM mode are: m/z 690.4 (double charge) → 782.3 (dp=85.08, ce= 40.88), m/z 690.4 (double charge) → 881.2 (dp=101.67, ce=39.06).
The results are shown in fig. 1 (the figure (A) is a reference substance, (B) is antelope horn, (C) is ox horn, (D) is yak horn, (E) is buffalo horn, and (F) is pig trotter horn), wherein the ion peak consistent with the characteristic peptide reference substance solution can be detected in antelope horn samples, and the characteristic peptide components in antelope horn can be specifically detected in other samples, so that the invention can be distinguished from other cuticle medicines.
Example 3 detection of characteristic Polypeptides in cornu Saigae Tataricae and cornu Naemorhedi
(1) Taking 20mg of antelope horn and goat horn samples, respectively adding 1ml of protein lysate [4%SDS,20mM DTT,50mM Tris-HCl (pH=8.8) ], homogenizing for 3 times, centrifuging at 12000rpm for 20min each time for 60s, measuring protein content by BCA, taking 1000 mug protein, adding 25 mug of 0.2M IAA solution, standing for 30min in a dark place, adding 5.5V of 80% cold acetone solution, fully mixing, and standing at-20 ℃ for freezing and precipitating overnight.
(2) Respectively taking acetone precipitation samples in the step (1), centrifuging at 12000rpm for 20min, discarding the supernatant, washing the precipitate once with 80% cold acetone, volatilizing the precipitate, adding 200 μl of 8M urea solution for re-dissolution, sufficiently shaking, adding 1400 μl of Tris-HCl buffer solution after the precipitate is completely dissolved to reduce the urea concentration to below 1M, respectively adding 0.1 μg/μl of trypsin 40 μl into each corner sample, and incubating for 12h in a constant-temperature water bath at 37 ℃ to obtain each corner enzymatic hydrolysate.
(3) The purified characteristic peptide of example 1 was prepared as a control solution at a concentration of 200ng/ml in 1ml, and 5. Mu.l of 0.2M IAA solution was added thereto and left in a dark place for 30min.
(4) Detecting the corner enzymolysis liquid in the step (2) and the characteristic peptide reference substance solution treated by IAA by adopting a liquid chromatography-mass spectrometer, wherein the liquid phase condition is as follows: the chromatographic column is 1.7 mu mWaters C column (2.1 mu m multiplied by 50 mm), the loading amount is 10 mu l, the flow rate is 0.3ml/min, the mobile phase A is 0.1% formic acid, the mobile phase B is 0.1% formic acid acetonitrile, the linear gradient elution is carried out for 0-3.5 min, 5-20% B, the linear gradient elution is carried out for 3.5-4 min, 20-5% B, the isocratic elution is carried out for 4-6 min and 5% B. Triple quadrupole mass spectrometry is adopted, and mass spectrometry parameters are as follows: the temperature of the ion source is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60psi; ion source gas 2, 60psi. The ion pairs for MRM mode are: m/z 690.4 (double charge) → 782.3 (dp=85.08, ce= 40.88), m/z 690.4 (double charge) → 881.2 (dp=101.67, ce=39.06).
The results are shown in fig. 2 (the graph (A) is a reference substance, (B) is antelope horn, (C) is goat horn), ion peaks consistent with the characteristic peptide reference substance solution can be detected in the antelope horn sample, and no detection is detected in the goat horn sample, so that the characteristic peptide components in the antelope horn can be specifically detected, and the characteristic peptide components can be distinguished from the goat horn.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> University of Nanjing traditional Chinese medicine
<120> Antelope horn characteristic peptide fragment and detection method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
Leu Lys Gln Glu Val Asn Cys Ala Tyr Val Arg
1 5 10
Claims (4)
1. A characteristic peptide fragment of antelope horn, which is characterized in that the characteristic peptide is as follows:
Leu-Lys-Gln-Glu-Val-Asn-Cys-Ala-Tyr-Val-Arg。
2. the method for detecting the characteristic peptide of the antelope horn is characterized by comprising the following steps of:
(1) Preparing the antelope horn characteristic peptide of claim 1 into a reference substance solution;
(2) After an animal medicine sample of the horn class to be detected is subjected to enzyme digestion by trypsin, respectively injecting enzymolysis liquid and the antelope horn characteristic peptide reference substance solution in the step (1) into a liquid-mass spectrometer, and adopting a multi-reaction monitoring mode by taking the antelope horn characteristic peptide as a reference, wherein the selected multi-reaction monitoring mode conditions are as follows: m/z 690.4 double charge→782.3, dp=85.08, ce= 40.88, m/z 690.4 double charge→881.2, dp=101.67, ce=39.06; if the ion pair is detected, the retention time of the ions is consistent with that of the antelope horn characteristic peptide reference substance, and the sub-ions of the ion pair are consistent with those of the reference substance, the sample to be detected contains antelope horn components;
the enzyme digestion method comprises the following steps: taking a sample 20 mg of a horn-like animal medicine to be detected, adding 1ml protein lysate, homogenizing for 3 times, centrifuging, taking supernatant, taking 1000 mug protein, adding 0.2M IAA solution, placing in a dark place, adding cold acetone solution, fully and uniformly mixing, refrigerating, and precipitating overnight;
Centrifuging an acetone precipitate sample, discarding supernatant, washing the precipitate once with cold acetone, volatilizing the precipitate, adding urea solution for re-dissolution, sufficiently shaking, adding Tris-HCl buffer solution after the precipitate is completely dissolved, respectively adding trypsin into each corner sample, and incubating in a constant-temperature water bath to obtain the acetone precipitate;
The liquid phase condition that the LC-MS detected is: the chromatographic column is a 1.7 mu m Waters C 18 column, the specification is 2.1 mu m multiplied by 50mm, the loading amount is 10 mu l, the flow rate is 0.3 ml/min, the mobile phase A is 0.1% formic acid, the mobile phase B is 0.1% acetonitrile formate, the linear gradient elution is carried out for 0-3.5 min and 5-20% B, the linear gradient elution is carried out for 3.5-4 min and 20-5% B, the isocratic elution is carried out for 4-6 min and 5% B; triple quadrupole mass spectrometry is adopted, and mass spectrometry parameters are as follows: the temperature of the ion source is 500 ℃; ionization voltage 5500V; the desolventizing temperature is 500 ℃; ion source gas 1, 60 psi; ion source gas 2, 60 psi; the ion pairs for MRM mode are: m/z 690.4 double charge → 782.3, dp=85.08, ce= 40.88, m/z 690.4 double charge → 881.2, dp=101.67, ce=39.06.
3. The method for detecting the characteristic peptide of antelope horn according to claim 2, wherein the enzyme digestion method is carried out as follows: taking a sample 20 mg of a to-be-detected horn-like animal medicine, adding 1ml protein lysate, adding grinding beads, homogenizing for 3 times, centrifuging at 12000 rpm for 20min each time for 60 s, taking 1000 mug of protein, adding 25 mug of 0.2M IAA solution, placing in a dark place for 30min, adding 5.5V Sample of percent cold acetone solution, fully mixing, and standing at-20 ℃ for freezing and precipitating overnight; the pH value of the protein lysate is 8.8, and the protein lysate comprises 4% sodium dodecyl sulfate, 20 mM dithiothreitol and 50 mM Tris-HCl;
Taking an acetone precipitate sample, centrifuging the acetone precipitate sample by 12000 rpm for 20 min, discarding the supernatant, washing the precipitate once by 80% cold acetone, volatilizing the precipitate, adding 200 μl of 8M urea solution for redissolution, sufficiently shaking, adding 1400 μl of Tris-HCl buffer solution after the precipitate is completely dissolved, reducing the urea concentration to below 1M, respectively adding 0.1 μg/μl of trypsin 40 μl into each corner sample, and incubating in a water bath at 37 ℃ for 12 h.
4. The method for detecting characteristic peptide of cornu Saigae Tataricae according to claim 2, wherein the horn-like animal medicine comprises cornu Saigae Tataricae, cornu Naemorhedi, cornu Bubali, ungula Sus domestica.
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