A characteristic polypeptide and its use

Abstract

The present application relates to a common characteristic polypeptide of Tridacnophorus, Tridacnophorus longirostris and Enneacanthus and its application and a method for identifying Tridacnophorus, Tridacnophorus longirostris and Enneacanthus, belonging to the technical field of biological technology detection.The present application provides a common characteristic polypeptide of Tridacnophorus, Tridacnophorus longirostris and Enneacanthus, and the amino acid sequence of the characteristic polypeptide is shown as SEQ ID NO.1.The characteristic polypeptide provided by the present application has excellent specificity and stability for Tridacnophorus, Tridacnophorus longirostris and Enneacanthus, is highly specific, can be used for identifying Tridacnophorus, Tridacnophorus longirostris and Enneacanthus, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology detection technology, and particularly relates to a common characteristic polypeptide of Trachyrhamphus serratus, Trachyrhamphus longirostris and Cirrhitichthys falco and application thereof and a method for identifying Trachyrhamphus serratus, Trachyrhamphus longirostris and Cirrhitichthys falco. BACKGROUND

[0002] Trachyrhamphus serratus is an important animal medicinal material in China, which has the effects of warming kidney and strengthening yang, resolving stasis and relieving swelling, and has a long history of use. The original sources are various. The Chinese Pharmacopoeia stipulates that the medicinal source of Trachyrhamphus serratus is the dried body of Trachyrhamphus serratus (Gray), Trachyrhamphus serratus (Gray) or Trachyrhamphus serratus (Gray). Solenognathus hardwickii Syngnathoides biaculeatus Syngnathusa cusLinnaeus

[0003] As a tonic medicinal material, the market demand for Trachyrhamphus serratus increases year by year. Since Trachyrhamphus serratus cannot be effectively artificially bred at present, the wild resources of Trachyrhamphus serratus are decreasing day by day, and many other species are mixed or inferior products are used instead. At present, the proportion of Trachyrhamphus serratus of different sources not included in the Chinese Pharmacopoeia increases year by year, leading to the confusion of the market circulation of Trachyrhamphus serratus. Trachyrhamphus serratus, Trachyrhamphus longirostris and Cirrhitichthys falco are common mixed species on the market due to large fishing quantity and low price. In addition, the three species have similar appearance and are often mixed and sold on the market.

[0004] At present, the methods for identifying and quality controlling Trachyrhamphus serratus mainly include characteristic identification, HPLC fingerprint method and molecular biology method. The characteristic identification method generally requires that the individual of Trachyrhamphus serratus be complete, and the identifier needs to have rich experience, so the method has a certain subjectivity. The HPLC fingerprint method has low specificity. The molecular biology method has high specificity, but is limited by the preservation of genetic material in the sample. For Trachyrhamphus serratus wine, processed products, extracts, insect-eaten and mildewed products and mixed samples of multiple individuals, it is difficult to extract DNA and conduct experiments. In recent years, more and more methods and standards use characteristic peptides to identify or quantitatively study natural medicines. The proteins contained in each species have differences in individual amino acids, which can be cut off by appropriate proteases as polypeptide segments with identification significance, i.e. characteristic peptides. The detection of characteristic peptides generally uses mass spectrometry, which does not need to consider the activity of proteins, and the preparation method is simple and the properties of peptides are relatively stable. The existing identification methods of Trachyrhamphus serratus have many limitations and cannot be applied to Trachyrhamphus serratus powder, processed products and extracts. The characteristic peptides in Trachyrhamphus serratus protein have the potential to be used as index components for the identification of Trachyrhamphus serratus. At present, the protein sequence information of Trachyrhamphus serratus, Trachyrhamphus longirostris and Cirrhitichthys falco has not been collected in each database, so it is impossible to obtain differential peptides by protein sequence comparison, and therefore there is no related record of characteristic polypeptides of Trachyrhamphus serratus, Trachyrhamphus longirostris and Cirrhitichthys falco. SUMMARY ​​​

[0005] The present application aims to provide a common characteristic polypeptide of Lollyfish, Long Lollyfish and Cirrhitichthys falco and its application and a method for identifying Lollyfish, Long Lollyfish and Cirrhitichthys falco. The characteristic polypeptide provided by the present application has excellent specificity and stability for Lollyfish, Long Lollyfish and Cirrhitichthys falco, and strong specificity, and can be used for species identification of Lollyfish medicinal materials, specifically Lollyfish, Long Lollyfish and Cirrhitichthys falco, and has good application prospect.

[0006] The present application provides a common characteristic polypeptide of Lollyfish, Long Lollyfish and Cirrhitichthys falco, and the amino acid sequence of the characteristic polypeptide is shown in SEQ ID NO. 1.

[0007] The present application also provides the application of the characteristic polypeptide in the above technical solution in identifying Lollyfish, Long Lollyfish and Cirrhitichthys falco.

[0008] Preferably, the identification is carried out using mass spectrometry, and the detection ion pair includes quantitative ions with a mass-to-charge ratio of m / z 719.1→329.2 and qualitative ions with a mass-to-charge ratio of m / z 719.1→797.4.

[0009] The present application also provides a method for identifying Lollyfish, Long Lollyfish and Cirrhitichthys falco, using the characteristic polypeptide shown in SEQ ID NO. 1 as a control.

[0010] The present application also provides a method for identifying Lollyfish, Long Lollyfish and Cirrhitichthys falco based on the characteristic polypeptide in the above technical solution, comprising the following steps:

[0011] (1) Preparation of test solution: mix the pretreated Lollyfish extract or pretreated Lollyfish powder extract with trypsin to obtain a mixed solution, mix the mixed solution with an ammonium bicarbonate aqueous solution, filter after enzymolysis, take the filtrate, and obtain a test solution;

[0012] (2) Preparation of control solution: use the common characteristic polypeptide of Lollyfish, Long Lollyfish and Cirrhitichthys falco in the above technical solution as a control, dissolve in water to obtain a control solution;

[0013] (3) Detection analysis: use triple quadrupole mass spectrometry for detection analysis;

[0014] There is no time sequence limitation between steps (1) and (2).

[0015] Preferably, in step (1), the preparation method of the pretreated sea dragon extract includes the following steps: sea dragon is crushed, mixed with water, and decocted three times, with decoction times of 4h, 3h and 2h respectively, the decoction liquid is combined, diluted with an ammonium bicarbonate aqueous solution, and a pretreated sea dragon extract is obtained;

[0016] The preparation method of the pretreated sea dragon powder extract includes the following steps: sea dragon powder is mixed with water and decocted three times, with decoction times of 4h, 3h and 2h respectively, the decoction liquid is combined, diluted with an ammonium bicarbonate aqueous solution, and a pretreated sea dragon powder extract is obtained.

[0017] Preferably, the trypsin is added in the form of a trypsin aqueous solution, and the mass concentration of the trypsin in the trypsin aqueous solution is 1mg / ml.

[0018] Preferably, the mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.

[0019] Preferably, the enzymolysis condition is 37℃ for 2h.

[0020] Preferably, the triple quadrupole mass spectrometry uses 0.1% formic acid acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, and gradient elution: 0~3min, 5%A→8%A; the flow rate is 0.5ml / min.

[0021] Preferably, the triple quadrupole mass spectrometry uses a mass spectrometry detector, electrospray positive ion mode, and multiple reaction monitoring, and the quantitative ion of SEQ ID NO. 1 is selected as m / z 719.1→329.2, and m / z 719.1→797.4 is selected as the qualitative ion as a detection ion pair.

[0022] The application provides a common characteristic polypeptide of Lophius litulon, Lophius litulon and Syngnathus groenblawdii. The characteristic polypeptide provided by the application is used as an index component for identifying Lophius litulon, Lophius litulon and Syngnathus groenblawdii, and can be distinguished from sea dragons of other species, and lays a foundation for developing an identification method for Lophius litulon, Lophius litulon and Syngnathus groenblawdii and related products. Specifically, the characteristic polypeptide has the following beneficial effects:

[0023] (1) The application cuts out a differential polypeptide segment in a protein sequence, and detects the polypeptide segment by using mass spectrometry, so that the operation is simple and fast, the determination result is stable, the sea dragon sample is not disturbed by the form and processing process, and the blank of a detection method for Lophius litulon, Lophius litulon and Syngnathus groenblawdii is filled in the identification of the species and the sea dragon product;

[0024] (2) The feature polypeptide and detection method provided by the application provide a reference for searching for characteristic peptide segments in related species lacking in databases;

[0025] (3) The feature polypeptide provided by the application has excellent specificity and stability for Trachyrhamphus serratus, Trachyrhamphus longirostris and Haliichthys taeniophorus, has strong specificity, and can be used for identification of Trachyrhamphus serratus, Trachyrhamphus longirostris and Haliichthys taeniophorus medicinal materials, and has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] The drawing is a specificity map of the common feature peptide SEQ ID NO. 1 of Trachyrhamphus serratus, Trachyrhamphus longirostris and Haliichthys taeniophorus provided by the application;

[0027] Among them, Figure 1 : SEQ ID NO. 1 feature peptide control; Figure 2 : Trachyrhamphus longirostris sample; Figure 3 : Haliichthys taeniophorus sample; Figure 4 : Trachyrhamphus serratus sample; Figure 5 : Dendrochirus zebra sample; Figure 6 : Syngnathus acus sample; Figure 7 : Dendrochirus zebra sample; Figure 8 : Syngnathus schlegeli sample; Figure 9 : Dendrochirus biocellatus sample. DETAILED DESCRIPTION

[0028] The application provides a common feature polypeptide of Trachyrhamphus serratus, Trachyrhamphus longirostris and Haliichthys taeniophorus, and the amino acid sequence of the feature polypeptide is as shown in SEQ ID NO. 1: MGPAGPAGIVGPAGPAGPAGKDGPR, the application provides that the water extract of different species of seahorses such as Trachyrhamphus serratus, Trachyrhamphus longirostris, Haliichthys taeniophorus, Dendrochirus zebra and Syngnathus schlegeli is subjected to trypsin enzymolysis, and then analyzed by using nanoliter liquid chromatography-high resolution mass spectrometry to obtain a seahorse protein full peptide mass spectrum; the mass spectrum data is subjected to chemometrics analysis, and then the specificity of the experimental results is verified by using a triple quadrupole mass spectrometer; finally, one characteristic ion pair is provided, corresponding to one characteristic peptide segment, and the specificity verification result is good.

[0029] The application also provides application of the feature polypeptide in identification of Trachyrhamphus serratus, Trachyrhamphus longirostris and Haliichthys taeniophorus.

[0030] In the application, the identification is performed by using mass spectrometry, and the detection ion pair comprises: quantitative ions with a mass-to-charge ratio of m / z 719.1→329.2 of SEQ ID NO. 1, and qualitative ions with a mass-to-charge ratio of m / z 719.1→797.4.

[0031] The application further provides a method for identifying H. crassicaudatus, H. longissimus and H. guttulatus based on the characteristic polypeptide, comprising the following steps:

[0032] (1) mixing the pretreated liquid extract of H. crassicaudatus or the pretreated liquid extract of H. longissimus with trypsin to obtain a mixed solution, mixing the mixed solution with an ammonium bicarbonate aqueous solution, performing enzymolysis, filtering, taking the filtrate, and obtaining a test sample solution;

[0033] (2) dissolving the common characteristic peptide of H. crassicaudatus, H. longissimus and H. guttulatus as a control sample in an ammonium bicarbonate aqueous solution to obtain a control sample solution;

[0034] (3) performing detection analysis by using a triple quadrupole mass spectrometer;

[0035] The steps (1) or (2) are not limited in time sequence.

[0036] The method has the advantages that the difference polypeptide segments in the protein sequence are cut out, detection is performed by using a mass spectrometer, the operation is simple and fast, the determination result is stable, the method is not disturbed by the form and processing process of the H. crassicaudatus sample, the blank of the detection method of H. crassicaudatus, H. longissimus and H. guttulatus is filled, and the method provides a reference for finding the characteristic peptide segments in the near relative species lacking in the database.

[0037] The pre-processed sea dragon extract or the pre-processed sea dragon powder extract is mixed with trypsin respectively to obtain a mixed solution, the mixed solution is mixed with an ammonium bicarbonate aqueous solution, and after enzymolysis, filtration is performed, and the filtrate is obtained as a test solution. In the present application, the preparation method of the pre-processed sea dragon extract preferably comprises the following steps: the L. rudis, L. longissimus and H. gilberti powder is crushed and mixed with water, and then boiled three times, the boiling time is 4 h, 3 h and 2 h respectively, the boiling liquid is combined, and the pre-processed sea dragon extract is obtained by diluting with an ammonium bicarbonate aqueous solution. In the present application, 100 g of crushed sea dragon is preferably taken, and the boiling liquid is preferably a total of 500 mL. In the present application, the dilution is preferably 10-fold dilution. Specifically, 5 mL of the boiling liquid is preferably taken, placed in a 50 mL volumetric flask, diluted to the calibration mark with an ammonium bicarbonate aqueous solution, shaken uniformly, and the pre-processed sea dragon extract is obtained. In the present application, the preparation method of the pre-processed sea dragon powder extract preferably comprises the following steps: the L. rudis, L. longissimus and H. gilberti powder is mixed with water and boiled three times, the boiling time is 4 h, 3 h and 2 h respectively, and the pre-processed sea dragon powder extract is obtained by combining the boiling liquid. In the present application, 1 g of sea dragon powder is preferably taken, and the boiling liquid is preferably a total of 50 mL. Specifically, 5 mL of the boiling liquid is preferably taken, placed in a 50 mL volumetric flask, diluted to the calibration mark with an ammonium bicarbonate aqueous solution, shaken uniformly, and the pre-processed sea dragon powder extract is obtained. After obtaining the pre-processed sea dragon extract and the pre-processed sea dragon powder extract, 1-5 mL of the sea dragon extract or the sea dragon powder extract is preferably taken, 100 μL of a trypsin solution is preferably added, shaken uniformly, 50 mL of an ammonium bicarbonate aqueous solution is added, shaken uniformly, and then the enzyme is hydrolyzed after the flask is tightly sealed. In the present application, the hydrolysis conditions are preferably 37℃ for 2 h. After hydrolysis, the present application is preferably cooled, and more preferably cooled to room temperature, and the temperature range of the room temperature in the present application is preferably 10-30℃. After cooling, the present application is filtered, and the filtrate is taken as a test solution.

[0038] In the present application, the trypsin is added in the form of a trypsin aqueous solution, and the mass concentration of the trypsin in the trypsin aqueous solution is 1 mg / ml. In the present application, the mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.

[0039] The common characteristic peptides of the L. rudis, L. longissimus and H. gilberti are dissolved in water as a control solution. The source of the common characteristic peptides of the L. rudis, L. longissimus and H. gilberti is not particularly limited in the present application, and an artificial synthesis method can be used, for example, the common characteristic peptides of the L. rudis, L. longissimus and H. gilberti in the present application are synthesized by Nanjing Yuandai Biological Technology Co., Ltd. according to the specified amino acid sequence.

[0040] The application utilizes triple quadrupole mass spectrometry for detection and analysis. In the application, the triple quadrupole mass spectrometry preferably uses 0.1% formic acid acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, and gradient elution: 0~3min, 5%A→8%A; the flow rate is 0.5ml per minute. In the application, the triple quadrupole mass spectrometry preferably uses a mass spectrometry detector, an electrospray positive ion mode, and multiple reaction monitoring, with the quantitative ion of SEQ ID NO. 1 being m / z 719.1→329.2 and the qualitative ion being m / z 719.1→797.4.

[0041] The common characteristic polypeptide of the one kind of Ceratochrysis, long Ceratochrysis and Gymnocranius grandoculis and the method for identifying the Ceratochrysis, long Ceratochrysis and Gymnocranius grandoculis are further described in detail below in combination with specific embodiments. The technical scheme of the application includes but is not limited to the following embodiments.

[0042] Triple quadrupole mass spectrometry determination conditions

[0043] 0.1% formic acid acetonitrile is used as mobile phase A, 0.1% formic acid solution is used as mobile phase B, and gradient elution (0~3min, 5%A→8%A) is performed; the flow rate is 0.5ml per minute. A mass spectrometry detector is used, an electrospray positive ion mode (ESI+) is used, multiple reaction monitoring (MRM) is performed, the quantitative ion of SEQ ID NO. 1 is m / z 719.1→329.2, and the qualitative ion is m / z 719.1→797.4.

[0044] Embodiment 1: Finding of a common characteristic peptide segment of Ceratochrysis, long Ceratochrysis and Gymnocranius grandoculis

[0045] (1) Preparation of a test sample solution

[0046] Sea dragon water extract: 100g of a test sample sea dragon was crushed, put into a triangular flask, and boiled three times with water, 4h, 3h and 2h respectively, and the boiled liquids were combined to 500ml, concentrated to a liquid paste under micro boiling, transferred to a silica gel bowl, and dried in a 60℃ electric heating constant temperature air drying oven to a solid, to obtain a sea dragon water extract;

[0047] Test sample solution: 0.1g of the sea dragon water extract sample was weighed, 50ml of 1% ammonium bicarbonate aqueous solution was added, ultrasonic treatment was performed for 30min to dissolve, a microporous filter was used for filtration, 100ul of the filtrate was taken, 10ul of 1mg / ml trypsin aqueous solution was added, and enzymolysis was performed at 37℃ for 2h, and then taken out and cooled to room temperature, to obtain the test sample solution.

[0048] (2) Selection of characteristic ions and sequence prediction

[0049] The mass spectrometry data of the test solution after nanoliter liquid chromatography-high resolution mass spectrometry analysis was introduced into PEAKS 8.5 software for de novo sequencing and sequence prediction of all peptides in the sample, and the obtained results were analyzed. The peptides that were only detected in L. guttatus, L. longissimus and H. guttulatus and almost not detected in other species of Lophiocephalus were selected as parent ions, and their secondary spectra were analyzed one by one to select the sub-ions with better response. Through analysis, the mass spectrometry ion pair information m / z 719.1 (double charge) → 329.2, 797.4 representing the differences between L. guttatus, L. longissimus and H. guttulatus was obtained. Through further analysis, it was speculated that the ion corresponded to the common characteristic peptide sequence of L. guttatus, L. longissimus and H. guttulatus, and SEQ ID NO. 1 was MGPAGPAGIVGPAGPAGPAGKDGPR.

[0050] The common characteristic peptide control of L. guttatus, L. longissimus and H. guttulatus was synthesized according to the predicted amino acid sequence, and the control was detected at the same time as the test solution of L. guttatus, L. longissimus and H. guttulatus, and the retention time and secondary mass spectrometry information of the two were consistent, thereby confirming the correctness of the sequence.

[0051] Example 2: Specificity study of common characteristic peptide of L. guttatus, L. longissimus and H. guttulatus

[0052] All species of Lophiocephalus were detected by triple quadrupole mass spectrometry determination conditions, and the results showed that the common characteristic peptide chromatographic peak of L. guttatus, L. longissimus and H. guttulatus appeared in three batches of L. guttatus, three batches of L. longissimus and three batches of H. guttulatus, while the corresponding chromatographic peak did not appear in other species of Lophiocephalus. See Figures 1-9 . It is shown that the characteristic peptide is specific to L. guttatus, L. longissimus and H. guttulatus, and can be used as an index component to identify L. guttatus, L. longissimus and H. guttulatus.

[0053] Example 3: Application of common characteristic peptide of L. guttatus, L. longissimus and H. guttulatus in Lophiocephalus extract and Lophiocephalus powder

[0054] (1) Sample preparation

[0055] Preparation of the test solution of the extract of the sea dragon: 100 g of each of the test samples of the crude sea dragon, the long and thick sea dragon and the Syngnathus grahami was ground and placed in a triangular flask, and water was added for decoction three times, 4 h, 3 h and 2 h respectively. The decocted liquid was combined, and the total volume was 500 mL. 5 mL of the extract of the sea dragon was taken and placed in a 50 mL volumetric flask, and 1% aqueous ammonium bicarbonate solution was added to dilute to the calibration mark. After shaking, 1 mL was taken and placed in a 5 mL volumetric flask, 100 μL of 1 mg / mL trypsin aqueous solution was added, and the mixture was shaken. Then, 1% aqueous ammonium bicarbonate solution was added to dilute to the calibration mark. After shaking, the flask was tightly sealed, and the mixture was incubated at 37°C for 2 h. After cooling, the mixture was filtered, and the filtrate was taken as the test solution.

[0056] Preparation of the test solution of the extract of the sea dragon powder: 1 g of each of the test samples of the crude sea dragon powder, the long and thick sea dragon powder and the Syngnathus grahami powder was placed in a triangular flask, and water was added for decoction three times, 4 h, 3 h and 2 h respectively. The decocted liquid was combined, and the total volume was 50 mL. 1 mL of the decocted liquid was taken and placed in a 5 mL volumetric flask, and the preparation was continued from the step of "adding 100 μL of 1 mg / mL trypsin aqueous solution".

[0057] Preparation of the control solution of the common characteristic peptide segment of the crude sea dragon, the long and thick sea dragon and the Syngnathus grahami: 10 mg of the control sample of the common characteristic peptide of the crude sea dragon, the long and thick sea dragon and the Syngnathus grahami (SEQ ID NO. 1) was weighed and dissolved in a 50 mL volumetric flask, and the volume was made up to the calibration mark.

[0058] (2) Determination by using the determination conditions of the triple quadrupole mass spectrometer.

[0059] (3) Sample determination

[0060] The determination was performed on 10 batches of the extract of the crude sea dragon, 10 batches of the extract of the long and thick sea dragon, 10 batches of the extract of the Syngnathus grahami, 5 batches of the powder of the crude sea dragon, 5 batches of the powder of the long and thick sea dragon and 5 batches of the powder of the Syngnathus grahami. The common characteristic peptide of the crude sea dragon, the long and thick sea dragon and the Syngnathus grahami was detected in all the samples, indicating that the characteristic peptide can be used for the identification of the common components of the crude sea dragon, the long and thick sea dragon and the Syngnathus grahami in the sea dragon products.

[0061] Example 4: Four ion information with identification potential were screened from 362 candidate ions, and the triple quadrupole mass spectrometer was used for verification. Not all the ions can be used for the identification of the specific components of the crude sea dragon, the long and thick sea dragon and the Syngnathus grahami. The specificity of some ions was not good enough. Only the ion with the mass-to-charge ratio of 719.1 had good specificity. The specific results are shown in Table 1.

[0062] Table 1 Specificity verification results

[0063]

[0064] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A polypeptide sharing common characteristics with *Syngnathus scaberra*, *Syngnathus longibrachioides*, and *Syngnathus gerberis*, characterized in that, The amino acid sequence of the characteristic polypeptide is shown in SEQ ID NO.

1.

2. The application of the characteristic polypeptide according to claim 1 in the identification of *Syngonium gracilis*, *Syngonium longibrillius*, and *Syngonium gracilis*.

3. The application according to claim 2, characterized in that, The identification was performed using mass spectrometry. The ion pairs detected by SEQ ID NO.1 included quantitative ions with a mass-to-charge ratio of m / z 719.1→329.2 and qualitative ions with a mass-to-charge ratio of m / z 719.1→797.

4.

4. A method for identifying the rough-snout seadragon, the long rough-snout seadragon, and the gerbera, characterized in that, Includes the following steps: (1) Preparation of test solution: The pretreated sea dragon extract or the pretreated sea dragon powder extract is mixed with trypsin to obtain a mixture. The mixture is then mixed with ammonium bicarbonate aqueous solution, enzymatically hydrolyzed, filtered, and the filtrate is collected to obtain the test solution. (2) Preparation of reference solution: The common characteristic peptides of the sea dragon, the long sea dragon and the sea bream described in claim 1 are used as references, and water is added to dissolve them to obtain reference solutions; (3) Detection and analysis: Detection and analysis were performed using triple quadrupole mass spectrometry; There is no time order requirement between steps (1) and (2); In step (1), the preparation method of the pretreated sea dragon extract includes the following steps: crushing the sea dragon, mixing it with water and decocting it three times for 4h, 3h and 2h respectively, combining the decoctions, and diluting it with ammonium bicarbonate aqueous solution to obtain the pretreated sea dragon extract; The preparation process of the pretreated seahorse powder extract includes the following steps: coarse snout seahorse powder, long coarse snout seahorse powder, and Ge's seahorse powder are mixed with water and decocted three times, with decocting times of 4h, 3h and 2h respectively. The decoctions are combined and diluted with ammonium bicarbonate aqueous solution to obtain the pretreated seahorse powder extract. The trypsin is added in the form of an aqueous solution of trypsin, and the mass concentration of the trypsin in the aqueous solution of trypsin is 1 mg / ml; The ammonium bicarbonate aqueous solution contains 1% ammonium bicarbonate by mass; the enzymatic hydrolysis conditions are 37°C for 2 hours. The triple quadrupole mass spectrometry method uses 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, with gradient elution: 0–3 min, 5% A → 8% A; flow rate is 0.5 ml per minute. The triple quadrupole mass spectrometry method employs a mass spectrometer detector, electrospray positive ion mode, and multiple reaction monitoring. SEQ ID NO.1 selects quantitative ions with mass-to-charge ratios m / z 719.1→329.2 and qualitative ions with m / z 719.1→797.4 as detection ion pairs.

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