Group of cornu gorais characteristic polypeptides and method for detecting cornu gorais in sample by using cornu gorais characteristic polypeptides

Abstract

The invention discloses a group of cornu gorais characteristic polypeptides and a method for detecting cornu gorais in a sample by using the cornu gorais characteristic polypeptides. The cornu gorais polypeptide provided by the invention is selected from any one or more of SEQ ID NO.1-8. According to the application, eight cornu gorais characteristic polypeptides are screened, the eight characteristic polypeptides are good in specificity, cornu gorais or a prescription preparation thereof can be distinguished from multiple cornu gorais medicinal materials or prescription preparations thereof, and qualitative identification of cornu gorais traditional Chinese medicinal materials is facilitated. The invention also develops a method for quantitatively detecting the content of cornu gorais in traditional Chinese medicinal materials or formulated preparations thereof and the content of the cornu gorais based on the six cornu gorais characteristic polypeptides, and the method adopts simple pretreatment steps, shortens the sample pretreatment time, is high in sensitivity, good in stability and high in accuracy, lays a foundation for improving the quality standard of the cornu gorais, and has a wide application prospect. Meanwhile, a thought and a method are provided for quality control research of other horn traditional Chinese medicines.

Description

Technical Field

[0001] The present application relates to the field of analysis and inspection of traditional Chinese medicine, and in particular to a group of goat horn characteristic polypeptides and a method for detecting goat horns in samples using the same. Background Art

[0002] Goat horn, originally recorded in the Shennong Bencao Jing (Shennong Bencao Jing), is included in local TCM standards, including the 2019 edition of the Guangdong Provincial Standard for Traditional Chinese Medicine, the 2022 edition of the Shandong Provincial Standard for Traditional Chinese Medicine, the 2020 edition of the Gansu Provincial Standard for Traditional Chinese Medicine, and the 2019 edition of the Jilin Provincial Standard for Traditional Chinese Medicine. Goat horn has the effects of clearing heat and calming nerves, dispersing blood stasis, and relieving pain. Modern pharmacological research has demonstrated significant antipyretic, sedative, analgesic, and anticonvulsant pharmacological activities. Due to its similar biological activity and chemical composition to antelope horn, goat horn has become a popular alternative to antelope horn in situations where its use is restricted, leading to a significant increase in its usage.

[0003] The main chemical components of goat horn include proteins, peptides, amino acids, nucleosides, and trace elements. Proteins are primarily high-molecular structures, primarily keratin; peptides are soluble peptides released through nonspecific degradation of proteins after decoction. Currently, goat horn quality standards are incomplete. Only some local standards cover identification and impurity testing, but lack dedicated identification methods and content determination methods.

[0004] Literature reports on the study of proteins in horn-based traditional Chinese medicines such as goat horn, buffalo horn, and yak horn. SDS protein lysis buffer was used, and the reducing agent dithiothreitol was added to extract total protein. The protein was purified by acetone precipitation, and then trypsin was used to obtain a peptide mixture. High-resolution mass spectrometry data of the peptides were collected. Based on the mass spectrometry data of multiple batches of samples from different origins, combined with multivariate statistical analysis, bioinformatics, and mathematical set analysis, the characteristic antelope horn peptides LKQEVNCAYVR (Pep-S1) and GGVTCGGLTYSSTAGR (Pep-S2) and goat horn characteristic peptides GPSLAGVSGSASSIR (Pep-G1) and GGVACGGLTYSSTAGR (Pep-G2) were screened out. These peptides can be distinguished from buffalo horn, yak horn, ox horn, and pig hoof, and can be used as detection indicators for species differentiation of horn-based traditional Chinese medicines.

[0005] The preparation methods of test samples reported in previous literature often use protein lysis buffer combined with reduction method, reducing the disulfide bond (RSSR) in the protein structure to cysteine ​​(RSH), and then alkylating to block the free sulfhydryl group, so that the protein is completely denatured and kept in a reduced state, and the protein extraction is relatively sufficient, and then trypsin is enzymatically digested to obtain peptides for analysis. This method can obtain rich peptide information for characteristic peptide screening. However, this method may have the following problems: (1) Characteristic peptides may not be applied to the quality control of horn-type medicinal materials in prescription preparations: the traditional method of using horn-type Chinese medicine is to heat and reflux in water, and the extract is mainly water-soluble protein, which is different from the protein obtained by protein lysis buffer extraction method. The protein to which the screened peptides belong may not be extracted by decoction method; (2) Pretreatment is complicated and time-consuming: the pretreatment process includes protein extraction, alkylation, protein purification, resolubilization and enzymatic hydrolysis, which is time-consuming; (3) It is difficult to carry out quantification: in the disulfide bond reduction alkylation treatment, the alkylation reaction is incomplete or the reagent is seriously excessive, the amount of alkylation introduced is uncertain, and it is difficult to accurately quantify. Summary of the Invention

[0006] The object of the present invention is to provide a group of goat horn polypeptides.

[0007] The object of the present invention is to provide a method for detecting goat horns in a sample.

[0008] In order to solve the above technical problems, the first aspect of the present invention provides a group of goat horn polypeptides, wherein the goat horn characteristic polypeptides are selected from any one or more of SEQ ID NO.1-8.

[0009] A second aspect of the present invention provides a method for detecting goat horns in a sample, wherein the detection includes qualitative and / or quantitative detection, and the method comprises the steps of:

[0010] The sample is identified using any one or more goat horn characteristic polypeptides in SEQ ID NO. 1-8.

[0011] In some preferred embodiments, the method includes the step of identifying the sample using the goat horn characteristic peptides of SEQ ID NO. 1-8.

[0012] In some preferred embodiments, the method includes the step of detecting whether the sample to be tested contains any one, two, three, four, five, six, seven, eight or all nine goat horn characteristic polypeptides in SEQ ID NO.1-8; if so, it is determined that the sample contains goat horn, otherwise it is determined that the sample does not contain goat horn.

[0013] In some preferred embodiments, the method includes the steps of: using LC-MS / MS (preferably ultra-high performance liquid chromatography-triple quadrupole mass spectrometry) to qualitatively detect whether the sample to be tested contains any one, two, three, four, five, six, seven, eight or all nine goat horn characteristic polypeptides in SEQ ID NO.1-8.

[0014] In some preferred embodiments, the method includes the steps of: using LC-MS / MS (preferably ultra-high performance liquid chromatography-triple quadrupole mass spectrometry) to quantitatively detect the content of any one, two, three, four, five, or six goat horn characteristic polypeptides of SEQ ID NO. 1-4, 7, and 8 in the test sample.

[0015] In some preferred embodiments, the LC-MS / MS chromatographic conditions include:

[0016] Column: C18;

[0017] Mobile phase: A is 0.1% formic acid solution, B is acetonitrile;

[0018] Gradient elution program: 0-10.0 min, 2%-22% B; 10.0-17.0 min, 22%-25% B; 17.0-17.5 min, 25%-90% B; and / or

[0019] Flow rate 0.3mL / min.

[0020] In some preferred embodiments, the chromatographic conditions of the LC-MS / MS include: XSelect Peptide CSHC18 chromatographic column (2.1 mm × 100 mm, 2.5 μm); column temperature 30°C; mobile phase: A is 0.1% formic acid solution, B is acetonitrile; gradient elution program: 0-10.0 min, 2%-22% B; 10.0-17.0 min, 22%-25% B; 17.0-17.5 min, 25%-90% B; flow rate 0.3 mL / min; injection volume 2 μL.

[0021] In some preferred embodiments, the mass spectrometry conditions of the LC-MS / MS include: electrospray ionization (ESI) positive ion mode; ion source temperature of 200°C; capillary voltage of 4000V; drying gas (N2) flow rate of 15L / min; nebulizing gas pressure (N2) of 30psi; sheath gas (N2) temperature of 300°C; and / or, flow rate of 11L / min.

[0022] In some preferred embodiments, the mass spectrometry conditions of the LC-MS / MS include: using 620.8 m / z as a parent ion, using 1070.5 m / z and 535.3 m / z as product ions, wherein 535.3 m / z is a quantitative product ion for determining SEQ ID NO.1;

[0023] SEQ ID NO. 2 was determined using 710.9 m / z as the parent ion and 1007.5 m / z and 750.4 m / z as the daughter ions, with 1007.5 m / z being the quantitative daughter ion.

[0024] SEQ ID NO. 3 was determined using 625.8 m / z as the parent ion and 940.5 m / z and 359.2 m / z as the daughter ions, with 359.2 m / z being the quantitative daughter ion.

[0025] SEQ ID NO. 4 was determined using 468.8 m / z as the parent ion and 695.4 m / z and 594.3 m / z as the daughter ions, with 594.3 m / z being the quantitative daughter ion.

[0026] SEQ ID NO. 5 was determined using 705.9 m / z as the parent ion and 1134.6 m / z and 888.5 m / z as the daughter ions, with 1134.6 m / z being the quantitative daughter ion.

[0027] SEQ ID NO. 6 was determined using 509.8 m / z as the parent ion and 563.3 m / z and 464.2 m / z as the daughter ions, with 563.3 m / z being the quantitative daughter ion.

[0028] SEQ ID NO. 7 was determined using 575.8 m / z as the parent ion and 1022.5 m / z and 859.5 m / z as the daughter ions, wherein 1022.5 m / z was the quantitative daughter ion; and / or

[0029] SEQ ID NO. 8 was determined using 582.8 m / z as the parent ion, 707.3 m / z and 175.1 m / z as the daughter ions, with 175.1 m / z being the quantitative daughter ion.

[0030] In some preferred embodiments, the LC-MS / MS mass spectrometry conditions include: using 620.8 m / z as a parent ion, using 1070.5 m / z and 535.3 m / z as product ions, wherein 535.3 m / z is a quantitative product ion, a collision voltage of 380 V, and a collision energy of 20 eV to determine SEQ ID NO.1;

[0031] SEQ ID NO. 2 was determined using 710.9 m / z as the parent ion, 1007.5 m / z and 750.4 m / z as the daughter ions, with 1007.5 m / z being the quantitative daughter ion, a collision voltage of 380 V, and a collision energy of 20-24 eV;

[0032] SEQ ID NO. 3 was determined using 625.8 m / z as the parent ion, 940.5 m / z and 359.2 m / z as the product ions, with 359.2 m / z being the quantitative product ion, a collision voltage of 380 V, and a collision energy of 16-20 eV;

[0033] SEQ ID NO. 4 was determined using 468.8 m / z as the parent ion, 695.4 m / z and 594.3 m / z as the daughter ions, with 594.3 m / z being the quantitative daughter ion, a collision voltage of 380 V, and a collision energy of 12-16 eV;

[0034] SEQ ID NO. 5 was determined using 705.9 m / z as the parent ion, 1134.6 m / z and 888.5 m / z as the product ions, with 1134.6 m / z as the quantitative product ion, a collision voltage of 380 V, and a collision energy of 24-28 eV.

[0035] SEQ ID NO. 6 was determined using 509.8 m / z as the parent ion, 563.3 m / z and 464.2 m / z as the daughter ions, with 563.3 m / z as the quantitative daughter ion, a collision voltage of 380 V, and a collision energy of 16-20 eV;

[0036] SEQ ID NO. 7 was determined using 575.8 m / z as the parent ion, 1022.5 m / z and 859.5 m / z as the daughter ions, wherein 1022.5 m / z was the quantitative daughter ion, a collision voltage of 380 V, and a collision energy of 24-28 eV; and / or

[0037] SEQ ID NO. 8 was determined using 582.8 m / z as the parent ion, 707.3 m / z and 175.1 m / z as the daughter ions, with 175.1 m / z being the quantitative daughter ion, a collision voltage of 380 V, and a collision energy of 32 eV.

[0038] In some preferred embodiments, the method comprises the steps of preparing a test solution using a 1% ammonium bicarbonate solution and a trypsin solution.

[0039] In some preferred embodiments, a 1% ammonium bicarbonate solution is used to prepare the reference solution.

[0040] In some preferred embodiments, the test solution is subjected to ultrasonic enzymatic hydrolysis and then directly detected.

[0041] In some preferred embodiments, the ultrasonic enzymatic hydrolysis is performed for no less than 6 hours.

[0042] In some preferred embodiments, the ultrasonic enzymatic hydrolysis lasts for 6-10 hours.

[0043] In some preferred embodiments, a non-glass container is used to place the sample solution for ultrasonic enzymatic hydrolysis.

[0044] In some preferred embodiments, a polypropylene container is used to place the test solution for ultrasonic enzymatic hydrolysis.

[0045] In some preferred embodiments, the amount of trypsin in the ultrasonic enzymatic hydrolysis step is about 0.04 mg / mL.

[0046] In some preferred embodiments, the ultrasonic enzymatic hydrolysis comprises the steps of: placing the test sample solution in a polypropylene container, and ultrasonically treating the solution in a water bath at 35° C. to 45° C. for 6 hours.

[0047] In some preferred embodiments, the method can accurately measure goat horns in the presence of horn-based medicinal materials other than sheep horns (such as yellow cattle horns, yak horns, and buffalo horns).

[0048] In some preferred embodiments, the method includes detecting whether the sample to be tested contains SEQ ID NO. 5, and the method can accurately detect goat horn in the presence of horn-based medicinal materials including sheep horn.

[0049] In some preferred embodiments, the sample is a Chinese medicinal material or a formulated preparation thereof.

[0050] In some preferred embodiments, the sample is a horn-type medicinal material or a formulated preparation thereof.

[0051] Based on the prior art, the present invention has at least the following advantages:

[0052] The present invention screened eight peptides characteristic of goat horn. These eight peptides possess high specificity and can distinguish goat horn or its formulations from a variety of horn-derived medicinal materials or their formulations, facilitating the qualitative identification of goat horn-derived Chinese medicinal materials. The present invention also developed a method for quantitatively detecting the goat horn content in Chinese medicinal materials or their formulations based on six of these peptides. This method utilizes simple pretreatment steps, shortens sample pretreatment time, and exhibits high sensitivity, stability, and accuracy. This method lays the foundation for improving the quality standards of goat horn and provides ideas and methods for quality control research of other horn-derived Chinese medicinal materials.

[0053] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] One or more embodiments are exemplarily described by the figures in the corresponding drawings, and these exemplified descriptions do not constitute limitations on the embodiments.

[0055] Figure 1 1 is a data quality control diagram according to an embodiment of the present invention (a. molecular mass distribution of proteins; b. coverage of proteins; c. charge distribution; d. unique peptide segments).

[0056] Figure 2 This is a Venn diagram of the number of proteins and peptides according to the embodiments of the present invention (a. Venn diagram of polypeptides of sheep horns and cow horns; b. Venn diagram of proteins of sheep horns and cow horns; c. Venn diagram of polypeptides of goat horns and sheep horns; d. Venn diagram of proteins of goat horns and sheep horns).

[0057] Figure 3 1 is a principal component analysis score diagram according to an embodiment of the present invention (QC quality control sample, SYJ goat horn, MYJ sheep horn, SNJ buffalo horn, MNJ yak horn, HNJ cattle horn).

[0058] Figure 4 is a secondary mass spectrum of a representative characteristic peptide according to an embodiment of the present invention.

[0059] Figure 5 1 is a linear fitting graph of Orbitrap RT-QQQ RT according to an embodiment of the present invention.

[0060] Figure 6 1 is a diagram showing the results of enzymatic hydrolysis time investigation according to an embodiment of the present invention.

[0061] Figure 7 1 is a diagram showing the results of ultrasound time investigation according to an embodiment of the present invention.

[0062] Figure 8 1 is a diagram showing the results of ultrasonic container material inspection according to an embodiment of the present invention.

[0063] Figure 9 1 is a diagram showing the results of a material investigation of a liquid phase vial according to an embodiment of the present invention.

[0064] Figure 10 1 is a diagram showing the results of investigating enzyme dosage according to an embodiment of the present invention.

[0065] Figure 11 2 is a graph showing the results of a specificity investigation according to an embodiment of the present invention.

[0066] Figure 12 3 batches of representative sample maps according to the examples of the present invention. DETAILED DESCRIPTION

[0067] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0068] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of this application.

[0069] Example 1

[0070] 1. Sample Collection

[0071] Twenty-two batches of goat horn samples were collected from various production areas, including Inner Mongolia, Shandong, and Zhejiang. Additionally, samples of easily confused medicinal materials, such as sheep horn, buffalo horn, yak horn, and ox horn, were collected. The sample collection details are shown in Table 1. All samples were authenticated by the Shanghai Institute for Food and Drug Inspection.

[0072] Table 1 Sample information

[0073]

[0074]

[0075] 2. Protein and peptide analysis and characteristic peptide screening

[0076] 2.1 Preparation of enzymatic peptides

[0077] 2.1.1 Total protein extraction

[0078] Take 0.5 g of fine powder of this product, accurately weigh it, put it into a flat-bottom flask, accurately add 15 mL of water, reflux for 1 hour, cool, filter, use 8 mL of the filter residue, reflux for 1 hour, filter, combine the filtrate, put it into a 25 mL volumetric flask, add water to the scale line, and obtain a water-soluble total protein solution. Centrifuge at 15000 r / min for 2 minutes, take 2 mL of the supernatant for ultrafiltration, centrifuge at 15000 r / min for 30 minutes, turn the ultrafiltration tube upside down on the collection tube, centrifuge at 15000 r / min for 2 minutes, take the upper layer (about 45 μL), add to 0.2 mL of 1% ammonium bicarbonate solution to obtain the total protein extract.

[0079] 2.1.2 Trypsin digestion

[0080] Take the total protein extract containing 100 μg of protein, add trypsin solution at a ratio of 25:1 (w / w) (take trypsin and add 1% ammonium bicarbonate solution to make a solution containing 1 mg in 1 mL, prepare it just before use). Shake well and enzymatically hydrolyze for 18 hours. Adjust the pH to 2 with 10% trifluoroacetic acid to inactivate the enzyme. Desalt the enzymatic solution using a MonoASpin C18 column, blow dry with nitrogen, add 100 μL of 0.1% formic acid solution to reconstitute, centrifuge at 15000 r / min for 1 minute, and collect the supernatant into a liquid phase vial.

[0081] 2.1.3 Quality Control (QC) Sample Preparation

[0082] Take 3 batches of the above supernatant samples of goat horn samples, buffalo horn samples, cattle horn samples, sheep horn samples, and yak horn samples, 20 μL of each batch, and mix them as QC samples.

[0083] 2.2 Protein concentration determination

[0084] The concentration of the total protein extract solution was determined using the Micro BCA 23235 analytical kit. The experimental steps are as follows: a series of bovine serum albumin (BSA) reference solutions with a concentration range of 2.5 to 40 μg / mL were prepared with water, 150 μL of the total protein test solution and reference solution after sample dilution were taken in a 96-well plate, 150 μL of the kit working solution was added to each diluted total protein test solution and reference solution, mixed for 30 seconds, incubated at 37°C for 2 hours, and the absorbance was measured at a wavelength of 562 nm using an enzyme reader. The total protein concentration in the sample was read from the standard curve. The standard curve Y = 0.0175X-0.0159, and the correlation coefficient R 2 The sample determination results are shown in Table 2, and the protein content is about 0.10%.

[0085] Table 2 Protein content determination results

[0086]

[0087]

[0088] 2.3 Nanoflow liquid chromatography-electrostatic field orbital ion trap high-resolution mass spectrometry conditions

[0089] Chromatographic conditions: Liquid phase separation was performed using an UltiMate 3000RSLC nano system. The mixed peptide sample was adsorbed onto an Acclaim PepMap 100C18 trap column (75 μm ID × 20 mm, 3 μm, ), in Acclaim PepMap 100C18 analytical column (75μm ID×25cm, 2μm, ) for separation, the binary mobile phase system: A is 0.1% formic acid solution, B is 80% acetonitrile solution; the gradient conditions are as follows: 0-3 min, 2.5% B; 3-75 min, 2.5-35.0% B; 75-80 min, 35.0-99.0% B; 80-85 min, 99.0% B; 85-86 min, 99.0% B; 86-100 min, 99.0-2.5% B; the flow rate is 300 nL / min, and the injection volume is 1 μL.

[0090] Mass spectrometry conditions: An Orbitrap Fusion Lumos mass spectrometer was used, and the electrospray ionization source parameters were set as follows: scanning in positive ion mode, positive ion spray voltage of 2300 V, and ion transfer tube temperature of 275°C. The Orbitrap mass analyzer primary scan range was m / z 350–2000, with a resolution of 120,000 (FWHM m / z 400), and the data format was Profile. The multistage mass spectrometer scan had a resolution of 30,000 and a centroid data format. Data-dependent scanning (DDA) was performed using the "Top speed" algorithm. Dynamic exclusion parameters included a cycle time of 2 s and an exclusion duration of 60 s. The precursor ion was fragmented using high-energy collision-induced dissociation (HCD), with collisional dissociation induced at 30% normalized collision energy (NCE), and ion charges of 2–5. Data acquisition and processing were performed using Xcalibur 4.0 software.

[0091] 2.4 Protein and peptide sequence identification

[0092] Proteome Discoverer 2.5 software was used to analyze the raw data from the RAW files using mass spectrometry. The UniProt database (uniprotkb_Bovidae_2024_08_19) (492,907 protein sequences) downloaded from UniProt was used as the search library, and the Seuqest HT algorithm was used to process the MS2 mass spectra. Parameters were: trypsin, a maximum of two deletion sites, a minimum peptide length of 6 amino acids, a precursor mass tolerance of 10 ppm, and a fragment mass tolerance of 0.02 Da. Fixed modifications: None; variable modifications (peptide termini): Oxidation, 15.99 Da (M, P); Deamidated, 0.984 Da (N, Q); Formyl, 27.995 Da (K, S, T); and variable modifications (protein termini): Acetyl, 42.01 Da (N-terminus). The peptide matching error rate was determined using a target-decoy strategy combined with Percolator modeling of correct and incorrect matches, and data filtering was performed at the peptide matching level using a Percolator-calculated q-value threshold (0.01) to control false discoveries.

[0093] A total of 893 proteins and 4,926 peptides were identified across 43 sample batches and 10 quality control samples. This method identified 57 keratins, 5 keratin-related proteins, and 21 collagens. 23.4% of the peptides were attributed to keratins, and 9.7% to collagens.

[0094] Data quality control information can be found in Figure 1 Most peptide precursor ions carry two or three positive charges. Identified proteins often contain two to four unique peptides, with molecular weights deviating less than 10 ppm from their theoretical values, ensuring the accuracy of the identification results. Protein molecular masses typically range from 50 to 100 kDa, and protein coverage is 10% to 20%.

[0095] Venn diagram Figure 2 Goat horns and cow horns share 1,799 peptides, with 1,096 and 511 unique peptides, respectively. They also share 367 proteins, with 164 and 127 unique proteins, respectively. Goat horns and sheep horns are highly similar, with 2,323 peptides in common and 572 and 592 unique peptides, respectively. They also share 435 proteins, with 96 and 119 unique proteins, respectively.

[0096] 2.5 Characteristic peptide screening

[0097] 2.5.1 Principal Component Analysis

[0098] Three-dimensional peptide data, including retention time, mass-to-charge ratio, and peak intensity, were derived. Retention time and mass-to-charge ratio were used as parameters, and peak intensity was used as observation. Missing values ​​were filled with the minimum value of the data matrix. The raw data were normalized, and data with peak intensities greater than 30% RSD in QC samples were deleted to generate a data matrix for statistical analysis. Unsupervised principal component analysis (PCA) (PCA-X) was performed using SIMCA-P software (version 14.1, Umetrics, MKS Instruments Inc.) with the UV method for scaling to understand the overall distribution of the different samples.

[0099] The principal component analysis results are as follows Figure 3 As shown, the QC samples clustered near the origin, indicating that the analytical system was capable of acquiring stable and reliable data. Since MNJ6 and MNJ7 were outside the 95% confidence interval, they were excluded from subsequent data analysis. The cumulative variance contribution of the first two principal components of the established model was 35.2%. The score plot shows that sheep horns differed significantly from cattle horns, while goat horns differed less from sheep horns. The horns of buffalo, yak, and cattle showed relatively little variation, indicating significant variability among subfamilies.

[0100] 2.5.2 Preliminary screening of characteristic peptides

[0101] A characteristic peptide is a peptide sequence unique to a species that can be specifically distinguished from other species. Representative samples were selected for interspecies comparison and preliminary screening of candidate characteristic peptides for each sample was conducted. The screening principles were: (1) t-test p value < 0.05; (2) peak intensity average ratio > 100 or < 0.01; (3) the average peak intensity of the representative sample was higher than 107. A total of 216 peptides were initially screened from goat horn, sheep horn, buffalo horn, cattle horn, and yak horn.

[0102] 2.5.3 Verification of the specificity of characteristic peptides

[0103] Three batches of samples each from goat horn, sheep horn, buffalo horn, cattle horn, and yak horn were selected to verify the sequence and specificity of the characteristic peptides. The verification was performed using ultra-performance liquid chromatography-triple quadrupole mass spectrometry in multiple reaction detection mode.

[0104] Chromatographic conditions: The chromatographic column used was Waters CORTECS T3 C18 (2.7 μm, 2.1 mm × 100 mm), the binary mobile phase system was: A was 0.1% formic acid solution, B was acetonitrile, the gradient elution program was: 0-40 min, 5%-35% B, the column temperature was 30°C, the flow rate was 0.3 mL / min, and the injection volume was 5 μL.

[0105] Mass spectrometry conditions: electrospray ionization (ESI) positive ion mode; ion source temperature 200°C, capillary voltage 4000 V, drying gas (N2) flow rate 15 L / min; nebulizing gas pressure (N2) 30 psi; sheath gas (N2) temperature 300°C, flow rate 11 L / min; nozzle voltage: 500 V.

[0106] A dynamic multiple reaction monitoring (DMRM) method was constructed. According to the secondary high-resolution mass spectra of each candidate characteristic peptide, fragment ions with good response and clear attribution were selected as product ions. The high-resolution secondary mass spectra of each peptide ion are shown in Figure 4 The peak time of the characteristic peptide sequence in the QQQ mass spectrometer was predicted by QQQ RT-Orbitrap RT linear fitting, as follows: according to the retention time distribution in the Orbitrap data, 8 representative ions were selected from the goat horn sample for QQQ MRM scanning, the peak time was recorded, and the QQQ RT and Orbitrap RT were linearly fitted. The linear equation is shown in Figure 5 The peak times of the remaining characteristic peptides were calculated using the equation. Based on the deviation between the predicted and detected retention times, a Δt value of 4 minutes was set. This led to the development of a DMRM method to investigate the specificity of candidate characteristic peptides. Nine peptides were screened from goat horns with excellent specificity, as shown in Table 3.

[0107] Table 3 9 characteristic peptide sequences and mass spectrometry information from goat horns

[0108]

[0109]

[0110] 2.5.4 Verification of the Accuracy of Characteristic Peptides

[0111] To verify the accuracy of the characteristic peptide sequences, the selected peptides were synthesized by Jier Biochemical (Shanghai) Co., Ltd. based on the identified peptide amino acid sequences. The purity of each peptide was greater than 95%. Using an established LC-MS / MS method, comparisons of retention times, parent ion and daughter ion information between peptide standards, peptide matrix standards, and samples revealed that SYJ1-22 was unstable in solution, so this peptide was removed. Eight characteristic peptides from goat horn were found to be effective in distinguishing cattle horn from cattle horn, with SYJ1-23 being particularly effective in distinguishing goat horn from sheep horn. The validation results are shown in Table 4.

[0112] Table 4 Validation results of 8 peptides in goat horn

[0113]

[0114] 3. Establishment of a method for identifying and determining the content of goat horns based on characteristic peptides

[0115] 3.1 Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry conditions

[0116] Chromatographic conditions: XSelect Peptide CSH C18 column (2.1 mm × 100 mm, 2.5 μm); column temperature 30°C; mobile phase: A: 0.1% formic acid solution, B: acetonitrile; gradient elution program: 0-10.0 min, 2%-22% B; 10.0-17.0 min, 22%-25% B; 17.0-17.5 min, 25%-90% B; flow rate 0.3 mL / min; injection volume 2 μL.

[0117] Mass spectrometry conditions: electrospray ionization (ESI) positive ion mode; ion source temperature 200°C, capillary voltage 4000V, drying gas (N2) flow rate 15L / min; nebulizing gas pressure (N2) 30psi; sheath gas (N2) temperature 300°C, flow rate 11L / min; MRM parameter information for each peptide is shown in Table 5.

[0118] Table 5 MRM parameters of peptides

[0119]

[0120] *Quantitative ion

[0121] 3.2 Preparation of reference solution

[0122] Standard stock solution: Take an appropriate amount of goat horn characteristic polypeptide reference substance, accurately weigh it, and add 1% ammonium bicarbonate solution to prepare a reference substance stock solution containing 1 mg per 1 mL.

[0123] Reference solution for sample addition: Dilute the above goat horn standard stock solution with 1% ammonium bicarbonate solution and mix to prepare the reference solution for sample addition. The concentrations of SYJ1-10 are 840 ng / mL; SYJ1-11 is 22432 ng / mL; SYJ1-14 is 22251 ng / mL; SYJ1-21 is 600 ng / mL; SYJ1-23 is 1350 ng / mL; SYJ1-4 is 3915 ng / mL; SYJ1-6 is 49497 ng / mL; and SYJ1-7 is 57072 ng / mL.

[0124] Standard curve solution: Accurately measure an appropriate amount of standard stock solution and add 1% ammonium bicarbonate solution to prepare reference solutions with concentrations of 0.5, 1, 2, 5, 10, 25, 50, 100, 250, and 500 ng / mL, respectively, as standard curve solutions.

[0125] 3.3 Preparation of test solution

[0126] 3.3.1 Investigation of test sample preparation methods

[0127] Enzyme hydrolysis time investigation: Take about 0.25g of the fine powder of this product, weigh it accurately, place it in a stoppered centrifuge tube, accurately add 24ml of 1% ammonium bicarbonate solution, and then accurately add 1ml of trypsin solution (take trypsin and add 1% ammonium bicarbonate solution to make a solution containing 1mg per 1ml, prepare it freshly before use), weigh it, and investigate the enzymatic hydrolysis time under the following conditions:

[0128] (1) Ultrasonicate in a water bath at 35°C to 45°C (power 1580W, frequency 37kHz) for 6 hours. Allow to cool, then weigh the sample. Make up the lost weight with 1% ammonium bicarbonate solution, shake well, and centrifuge. Accurately measure 1 ml of the supernatant in a 10 ml volumetric flask, dilute to the mark with 1% ammonium bicarbonate solution, shake well, centrifuge, and collect the supernatant.

[0129] (2) Ultrasonicate in a water bath at 35°C to 45°C (power 1580W, frequency 37kHz) for 6 hours, place in a 37°C oven for enzymatic hydrolysis for 12 hours, and inject the sample. Let cool, then weigh again. Make up the lost weight with 1% ammonium bicarbonate solution, shake well, and centrifuge. Accurately measure 1 ml of the supernatant into a 10 ml volumetric flask, dilute to the mark with 1% ammonium bicarbonate solution, shake well, centrifuge, and remove the supernatant.

[0130] After experimental analysis, it was found that ultrasonic enzymatic hydrolysis for 6 hours and then 12 hours of enzymatic hydrolysis did not significantly increase the peptide concentration, so it was finally decided to inject the sample directly after ultrasonication. Figure 6 .

[0131] Ultrasonic Time Study: Approximately 0.25 g of fine powder of this product was accurately weighed and placed in a stoppered centrifuge tube. 24 ml of 1% ammonium bicarbonate solution was accurately added, followed by 1 ml of trypsin solution (trypsin was added to 1% ammonium bicarbonate solution to make a solution containing 1 mg per ml, prepared freshly just before use). The solution was weighed and ultrasonically treated in a water bath at 35°C to 45°C (power 1580 W, frequency 37 kHz) for 1 h, 3 h, 6 h, 9 h, and 12 h. The solution was cooled and weighed again. The weight was made up with 1% ammonium bicarbonate solution, shaken, and centrifuged. Accurately measure 1 ml of the supernatant in a 10 ml volumetric flask, diluted to the mark with 1% ammonium bicarbonate solution, shaken, centrifuged, and the supernatant was obtained.

[0132] The results showed that as the ultrasonic time increased, the peptide was completely extracted after 6 hours of ultrasonic treatment, and the response of the peptide decreased after 12 hours of ultrasonic treatment. The ultrasonic time was finally determined to be 6 hours. Figure 7 .

[0133] Ultrasonic container material inspection: Take about 0.25g of the fine powder of this product, weigh it accurately, and conduct ultrasonic container material inspection under the following conditions: (1) 50mL polypropylene centrifuge tube, (2) 100mL glass conical flask, and operate according to the test sample preparation method. The results show that the glass extraction container will reduce the concentration of polypeptide. Finally, it is determined that the extraction container is a 50mL polyethylene centrifuge tube, see Figure 8 .

[0134] Liquid phase vial material inspection Take an appropriate amount of the test solution and place it in a polypropylene liquid phase vial and a glass liquid phase vial, respectively, and inject the sample at 0h, 12h, and 24h. The results show that there is no significant difference in the test solution after being placed in a polypropylene liquid phase vial or a glass liquid phase vial for 24h. Considering daily use, it was finally decided to use a glass liquid phase vial. Figure 9 . Investigation of enzyme dosage: Take about 0.25 g of the fine powder of this product, weigh it accurately, put it in a stoppered centrifuge tube, accurately add an appropriate amount of 1% ammonium bicarbonate solution, and investigate the enzyme dosage under the following conditions: accurately add 0.25, 0.50, 1.00, and 1.50 mL of trypsin solution (take trypsin, add 1% ammonium bicarbonate solution to make a solution containing 1 mg per 1 ml, and make it fresh before use), add 1% ammonium bicarbonate solution to 25 mL, weigh it, and ultrasonically treat it in a water bath at 35℃~45℃ (power 1580W, frequency 37kHz) for 6 hours, let it cool, weigh it again, make up the lost weight with 1% ammonium bicarbonate solution, shake it well, centrifuge it, and take the supernatant. The results showed that the extraction was basically complete when the enzyme dosage was 1.00 mL, and only SYJ1-6 showed an upward trend when the enzyme dosage was increased. Considering the cost issue, the enzyme dosage was selected as 1.00 mL. Figure 10 .

[0135] 3.3.2 Determined method for preparing test solution

[0136] Accurately weigh approximately 0.25 g of this fine powder and place it in a stoppered centrifuge tube. Accurately add 24 ml of 1% ammonium bicarbonate solution, then accurately add 1 ml of trypsin solution (take trypsin and add 1% ammonium bicarbonate solution to a solution containing 1 mg per 1 ml, prepare freshly just before use). Weigh the solution and sonicate in a 35-45°C water bath (power 1580 W, frequency 37 kHz) for 6 hours. Let cool, reweigh the solution, make up the lost weight with 1% ammonium bicarbonate solution, shake well, and centrifuge. Accurately measure 1 ml of the supernatant in a 10 ml volumetric flask, dilute to the mark with 1% ammonium bicarbonate solution, shake well, centrifuge, and remove the supernatant.

[0137] 3.4 Methodological validation

[0138] 3.4.1 Specificity

[0139] Sheep horn, ox horn, yak horn, and buffalo horn were prepared according to the test solution preparation method. Samples were injected and analyzed. The ion chromatogram of the goat horn extraction showed 8 peptide peaks. In the ion chromatogram of the sheep horn extraction, in addition to the peptide SYJ1-23, there were 7 chromatographic peaks with the same retention time as the goat horn. For details, see Figure 11 .

[0140] 3.4.2 Linear Relationship

[0141] The standard curve solution was sampled and measured, and the quantification was carried out using the external standard method. The standard curve was drawn. The linear range and correlation coefficient results are shown in Table 6. Each peptide showed a good linear relationship within the corresponding concentration range.

[0142] Table 6 Linear relationships of 8 characteristic peptides from goat horns

[0143] peptides Linear Linear range (ng / mL) r SYJ1-10 y=2670.47x+87.48 0.5-10 0.999 SYJ1-11 y=944.56x-2438.33 10-250 0.999 SYJ1-14 y=2050.54x-640.50 1-25 0.998 SYJ1-21 y=3008.74x-170.33 0.5-10 0.999 SYJ1-23 y=1561.30x-892.90 1-25 0.997 SYJ1-4 y=3264.27x-677.39 2--50 0.998 SYJ1-6 y=613.42x-3310.10 25-500 0.998 SYJ1-7 y=606.86x-3148.20 25-500 0.999

[0144] 3.4.3 Precision

[0145] The reference solution (100 ng / mL) was injected six times, and the peak areas were recorded and the RSDs were calculated. The results are shown in Table 7. The peak area RSDs for peptides SYJ1-4 were 17.64%, indicating that the peptides were relatively unstable and should only be used for qualitative identification of goat horns. The peak area RSDs for the remaining peptides were all less than 9.0%.

[0146] Table 7 Precision investigation

[0147] Self-numbering 1 2 3 4 5 6 RSD (%) SYJ1-10 329966 343421 339660 337179 346243 344423 1.76 SYJ1-11 84123 94311 101239 101328 105068 105177 8.21 SYJ1-14 221897 243148 244779 245785 239578 244041 3.78 SYJ1-21 366776 367477 364487 372042 388661 361485 2.62 SYJ1-23 187997 188573 193901 195453 179337 184732 3.15 SYJ1-4 364851 353234 289610 285593 247623 239456 17.64 SYJ1-6 52772 55675 56143 57672 60030 60813 5.21 SYJ1-7 56472 57319 59338 59076 61657 60521 3.27

[0148] 3.4.4 Repeatability

[0149] 0.25g of goat horn sample No. 1 was prepared in sextuplicate. Solutions were prepared according to the test sample preparation method, and the content and RSD were calculated. The results are shown in Table 8. The RSD for sextuplicate repeatability was 3.5% to 5.8%, indicating good reproducibility.

[0150] Table 8 Repeatability study of 8 characteristic peptides from goat horns (μg / g)

[0151]

[0152]

[0153] 3.4.5 Accuracy

[0154] Take 0.125g of goat horn sample No. 1, accurately weigh it, and place it in a stoppered centrifuge tube. Make nine replicates, with three replicates forming one group. Accurately add 0.667mL, 0.833mL, and 1mL of the reference solution to each group, respectively. Accurately add 1mL of trypsin solution (take trypsin and add 1% ammonium bicarbonate solution to make a solution containing 1mg per 1mL, prepared freshly just before use). Supplement with 1% ammonium bicarbonate solution to 25mL, weigh the sample, and sonicate in a water bath at 35°C to 45°C (power 1580W, frequency 37kHz) for 6 hours. Let cool, weigh again, make up the lost weight with 1% ammonium bicarbonate solution, shake well, and centrifuge. Accurately measure 1mL of the supernatant into a 10mL volumetric flask, dilute to the mark with 1% ammonium bicarbonate solution, shake well, centrifuge, and remove the supernatant.

[0155] The results are shown in Table 9. The average recovery rate of the eight characteristic peptides was 89.7% to 147.0%, and the RSD of nine replicates was 7.1% to 12.0%. Among them, the recovery rate of SYJ1-23 was relatively high, which was used as a qualitative detection indicator; the average recovery rate of the other seven characteristic peptides was 89.7% to 109.8%, which was used for content determination.

[0156] Table 9 Accuracy assessment of 8 characteristic peptides

[0157]

[0158]

[0159]

[0160] 3.4.6 Stability

[0161] A test solution of goat horn sample 1 was prepared and allowed to stand at room temperature. Samples were injected and analyzed at 0, 4, 8, 12, 16, and 24 hours. Peak areas were recorded and RSDs were calculated. The results are shown in Table 10. The RSDs for peak areas for all eight peptides were less than 7.0% within 24 hours, demonstrating substantial stability over that time.

[0162] Table 10 Stability of 8 characteristic peptides

[0163] Self-numbering 0h 8h 12h 16h 20h 24h RSD (%) SYJ1-10 15045 14171 15792 16250 14446 15770 5.42 SYJ1-11 134543 137339 144865 142506 140506 138032 2.68 SYJ1-14 28653 30467 30069 32226 31740 30723 4.14 SYJ1-21 9908 9518 9976 10505 10969 11180 6.30 SYJ1-23 10392 11412 11273 11551 11259 11980 4.62 SYJ1-4 154522 148108 144271 132354 146355 128697 6.93 SYJ1-6 253910 272212 260600 275179 279325 283574 4.20 SYJ1-7 219669 237551 239467 224750 219442 235155 3.98

[0164] 3.5 Sample determination

[0165] The above method was used to analyze 22 batches of goat horn samples. The results showed that all of them presented characteristic peaks of 8 polypeptides. The content results are shown in Table 1-16. The average content of SYJ1-10 measured in 22 batches of goat horns was 0.00049%; SYJ1-11 was 0.011%; SYJ1-14 was 0.0014%; SYJ1-21 was 0.00031%; SYJ1-6 was 0.027%; and SYJ1-7 was 0.020%.

[0166] Representative goat horn sample atlas Figure 12 , all showed 8 chromatographic peaks with the same retention time as the reference substance.

[0167] Table 11 Content of 6 characteristic peptides in 22 samples (%)

[0168]

[0169]

[0170] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present application, and that in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present application.

[0171] Sequence Listing:

[0172] Serial number Peptide number Amino acid sequence SEQ ID NO.1 SYJ1-10 AVTGFDDPFSGK SEQ ID NO.2 SYJ1-11 GGVTVSGISSSSNIR SEQ ID NO.3 SYJ1-14 YFSGNPIIPSR SEQ ID NO.4 SYJ1-21 AAVTGFWGK SEQ ID NO.5 SYJ1-23 YLDMDSIIAEIK (deamidation at position 3) SEQ ID NO.6 SYJ1-4 QLDVVSSDR SEQ ID NO.7 SYJ1-6 KYEEEIALR SEQ ID NO.8 SYJ1-7 RYEEEVALR

Claims

1. A group of goat horn polypeptides, characterized in that The goat horn characteristic polypeptide is selected from any one or more of SEQ ID NO.1-8.

2. A method for detecting goat horns in a sample, characterized in that: The detection includes qualitative and / or quantitative detection, and the method includes the steps of: using any one or more goat horn characteristic polypeptides in SEQ ID NO. 1-8 to identify the sample.

3. The method according to claim 2, characterized in that The method comprises the steps of: preparing a test solution by using a 1% ammonium bicarbonate solution and a trypsin solution.

4. The method according to claim 3, characterized in that The method comprises the steps of: taking a test sample solution, performing ultrasonic enzymatic hydrolysis, and then directly detecting.

5. The method according to claim 4, characterized in that The ultrasonic enzymatic hydrolysis lasts for 6-10 hours.

6. The method according to claim 4, characterized in that Use a non-glass container to place the test solution for the ultrasonic enzymatic hydrolysis.

7. The method according to claim 4, characterized in that The ultrasonic enzymatic hydrolysis comprises the following steps: placing a test sample solution in a polypropylene container, and ultrasonically treating the solution in a water bath at 35° C. to 45° C. for 6 hours.

8. The method according to claim 2, characterized in that The method can accurately measure goat horns in the presence of horn-based medicinal materials other than sheep horns (such as yellow cattle horns, yak horns, and buffalo horns); And / or, the method includes detecting whether the sample to be tested contains SEQ ID NO. 5, and the method can correctly detect goat horns in the presence of horn-based medicinal materials including sheep horns.

9. The method according to claim 2, characterized in that The method comprises the steps of: using LC-MS / MS (preferably ultra-high performance liquid chromatography-triple quadrupole mass spectrometry) to qualitatively detect whether the sample to be tested contains any one, two, three, four, five, six, seven, eight or all nine goat horn characteristic polypeptides in SEQ ID NOs. 1-8; And / or, the method includes the steps of: using LC-MS / MS (preferably ultra-high performance liquid chromatography-triple quadrupole mass spectrometry) to quantitatively detect the content of any one, two, three, four, five, or six goat horn characteristic polypeptides of SEQ ID NOs. 1-4, 7, and 8 in the test sample.

10. The method according to claim 9, characterized in that The mass spectrometry conditions of the LC-MS / MS include: using 620.8 m / z as a parent ion, using 1070.5 m / z and 535.3 m / z as product ions, wherein 535.3 m / z is a quantitative product ion for determining SEQ ID NO.1; SEQ ID NO. 2 was determined using 710.9 m / z as the parent ion and 1007.5 m / z and 750.4 m / z as the daughter ions, with 1007.5 m / z being the quantitative daughter ion. SEQ ID NO. 3 was determined using 625.8 m / z as the parent ion and 940.5 m / z and 359.2 m / z as the daughter ions, with 359.2 m / z being the quantitative daughter ion. SEQ ID NO. 4 was determined using 468.8 m / z as the parent ion and 695.4 m / z and 594.3 m / z as the daughter ions, with 594.3 m / z being the quantitative daughter ion. SEQ ID NO. 5 was determined using 705.9 m / z as the parent ion and 1134.6 m / z and 888.5 m / z as the daughter ions, with 1134.6 m / z being the quantitative daughter ion. SEQ ID NO. 6 was determined using 509.8 m / z as the parent ion and 563.3 m / z and 464.2 m / z as the daughter ions, with 563.3 m / z being the quantitative daughter ion. SEQ ID NO. 7 was determined using 575.8 m / z as the parent ion and 1022.5 m / z and 859.5 m / z as the daughter ions, wherein 1022.5 m / z was the quantitative daughter ion; and / or SEQ ID NO. 8 was determined using 582.8 m / z as the parent ion, 707.3 m / z and 175.1 m / z as the daughter ions, with 175.1 m / z being the quantitative daughter ion.

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