Characteristic polypeptide of Diaothias multispinata, its application and method for identifying Diaothias multispinata

Abstract

The present invention relates to a characteristic polypeptide of the multispined sea dragon, its application, and a method for identifying the multispined sea dragon, belonging to the field of biotechnology detection technology. The present invention provides two characteristic polypeptides unique to the multispined sea dragon, the amino acid sequences of the characteristic polypeptides being shown as SEQ ID NO.1 and SEQ ID NO.2. The characteristic polypeptides provided by the present invention have excellent specificity and stability for the multispined sea dragon, have high specificity, can be used for the identification of the multispined sea dragon, and have good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology detection technology, and in particular to two characteristic polypeptides of Diaothias multispinata and their applications, and a method for identifying Diaothias multispinata. Background Art

[0002] Sea dragon is an important animal medicinal material in my country, known for its kidney-warming, yang-strengthening, and swelling-reducing properties. It has a long history of medicinal use and numerous sources. The Chinese Pharmacopoeia defines sea dragon as the dried body of the Syngnathidae family members Solenognathus hardwickii (Gray), Syngnathoides biaculeatus (Bloch), or Syngnathusa cusLinnaeus.

[0003] As a tonic medicinal material, the market demand for sea dragons is increasing year by year. However, due to the current inability to effectively cultivate sea dragons in captivity, wild sea dragon resources are decreasing, and they are often confused with other species or sold as inferior products. Currently, the sales proportion of sea dragons from different sources that are not included in the Chinese Pharmacopoeia is increasing year by year, leading to chaotic circulation of sea dragon medicinal materials in the market. The multi-spined sea dragon and the multi-spined sea dragon are closely related species with similar names. It is the most common confusion with the multi-spined sea dragon and is often sold in the market as the authentic multi-spined sea dragon. The grade of commercially available sea dragon medicinal materials is mostly divided by the size of the sea dragon. Due to the large size of the multi-spined sea dragon, its market price is even higher than that of the authentic multi-spined sea dragon and other sea dragon products.

[0004] Currently, the main methods for identifying and controlling sea dragon species include trait identification, HPLC fingerprinting, and molecular biology. Trait identification generally requires intact sea dragon specimens and extensive experience, leading to a degree of subjectivity. HPLC fingerprinting offers low specificity, while molecular biology offers high specificity but is limited by the preservation of genetic material in the sample. DNA extraction from sea dragon medicinal liquors, processed products, extracts, insect-damaged and moldy specimens, and samples containing multiple individuals presents challenges. In recent years, a growing number of methods and standards have utilized signature peptides for the identification or quantification of natural medicinal products. Proteins from different species exhibit amino acid differences at specific sites. These differential sites can be extracted using appropriate proteases to create identifying peptides, known as signature peptides. Detection of signature peptides is typically achieved using mass spectrometry, which eliminates the need for protein activity, facilitates simple preparation, and provides relatively stable peptide properties. Existing methods for identifying sea dragons are limited by numerous factors, preventing their application in derivative products such as sea dragon powders, sea dragon medicinal wines, processed products, and extracts. Signature peptides from sea dragon proteins have the potential to serve as indicators for identifying sea dragon species. Currently, no databases contain protein sequence information related to the multispinous sea dragon, making it impossible to identify differential peptides through protein sequence comparison. Consequently, no records of its characteristic peptides exist. Summary of the Invention

[0005] The present invention aims to provide two characteristic polypeptides of the multispinous sea dragon, their applications, and methods for identifying the multispinous sea dragon. The characteristic polypeptides provided by the present invention have excellent specificity and stability for the multispinous sea dragon, with strong specificity. They can be used to identify sea dragon medicinal materials, specifically the multispinous sea dragon, and have good application prospects.

[0006] The present invention provides two characteristic polypeptides of Diaothias multispinata, and the amino acid sequences of the characteristic polypeptides are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0007] The present invention also provides the use of the characteristic polypeptide described in the above technical solution in identifying the multispinous sea dragon.

[0008] Furthermore, the use of SEQ ID NO.1 and SEQ ID NO.2 alone or together in identifying the multispinous sea dragon.

[0009] Preferably, the identification is performed using mass spectrometry, and the detected ion pairs include: a quantitative ion with a mass-to-charge ratio of m / z 676.3→418.2 for SEQ ID NO.1, and a qualitative ion with a mass-to-charge ratio of m / z 676.3→603.3; a quantitative ion with a mass-to-charge ratio of m / z 521.7→631.3 for SEQ ID NO.2, and a qualitative ion with a mass-to-charge ratio of m / z 521.7→517.2.

[0010] The present invention also provides a method for identifying the multispinous sea dragon, using the characteristic polypeptide shown in SEQ ID NO.1 and / or SEQ ID NO.2 as a reference substance.

[0011] The present invention also provides a method for identifying the multispinous sea dragon based on the characteristic polypeptide described in the above technical solution, comprising the following steps:

[0012] (1) Preparation of the test solution: The pretreated sea dragon extract or the pretreated sea dragon medicinal wine extract was mixed with trypsin to obtain a mixed solution, the mixed solution was mixed with an aqueous ammonium bicarbonate solution, and the mixture was enzymatically hydrolyzed and filtered to obtain the test solution;

[0013] (2) Preparation of reference solution: using the characteristic peptide of the multispinous sea dragon described in the above technical solution as a reference substance, dissolving it in water to obtain a reference solution;

[0014] (3) Detection and analysis: Detection and analysis were performed using triple quadrupole mass spectrometry;

[0015] There is no time sequence limitation between the steps (1) and (2).

[0016] Preferably, in step (1), the method for preparing the pretreated sea dragon extract comprises the following steps: crushing the sea dragon, mixing it with water and decocting it three times, with the decocting time being 4 hours, 3 hours and 2 hours respectively, combining the decoctions, and diluting them with an aqueous ammonium bicarbonate solution to obtain the pretreated sea dragon extract;

[0017] The preparation process of the pretreated sea dragon medicinal wine extract comprises the following steps: taking out the sea dragon from the sea dragon medicinal wine, drying at low temperature, decocting with water three times, the decocting time being 4 hours, 3 hours and 2 hours respectively, combining the decoctions, filtering, and taking the filtrate to obtain the pretreated sea dragon medicinal wine extract;

[0018] 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 1 mg / ml.

[0019] Preferably, the mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.

[0020] Preferably, the enzymatic hydrolysis condition is 37° C. for 2 h.

[0021] Preferably, 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, and performs gradient elution: 0-3 min, 5% A→8% A; the flow rate is 0.5 ml per minute.

[0022] Preferably, the triple quadrupole mass spectrometry uses a mass spectrometer detector in electrospray positive ion mode to perform multiple reaction monitoring, and SEQ ID NO.1 selects a quantitative ion with a mass-to-charge ratio of m / z 676.3→418.2 and a qualitative ion with a mass-to-charge ratio of m / z 676.3→603.3 as the detection ion pair, and SEQ ID NO.2 selects a quantitative ion with a mass-to-charge ratio of m / z 521.7→631.3 and a qualitative ion with a mass-to-charge ratio of m / z 521.7→517.2 as the detection ion pair.

[0023] The present invention provides two characteristic polypeptides of the multispinous sea dragon. The characteristic polypeptides provided by the present invention serve as indicator components for identifying the multispinous sea dragon and can distinguish it from other species of sea dragons, laying the foundation for the development of identification methods for the multispinous sea dragon and related products. Specifically, the characteristic polypeptides of the present invention have the following beneficial effects:

[0024] (1) The present invention extracts differential polypeptide segments from protein sequences and uses mass spectrometry for detection. The operation is simple and fast, the measurement results are stable, and the method is not affected by the morphology and processing of the sea dragon sample. This fills the gap in the identification of the multi-spined sea dragon species and the detection method of the multi-spined sea dragon in sea dragon products.

[0025] (2) The characteristic peptides and detection methods provided by the present invention provide a reference for finding characteristic peptides in closely related species that are lacking in the database;

[0026] (3) The characteristic polypeptide provided by the present invention has excellent specificity and stability for the multi-spined sea dragon, has strong specificity, can be used for the identification of the multi-spined sea dragon medicinal material, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Attachment Figure 1-9 The specificity map of the characteristic peptide SEQ ID NO.1 of the multispinous sea dragon provided by the present invention;

[0028] in: Figure 1 : SEQ ID NO.1 characteristic peptide control; Figure 2 : Sample of Multispined Sea Dragon; Figure 3 : Sample of Pseudohalodon; Figure 4 : Schlossosaurus sample; Figure 5 : Diao Hailong sample; Figure 6 : Sample of Baojia spear-nosed sea dragon; Figure 7 : Sample of Long Thick-snouted Sea Dragon; Figure 8 : Sample of Roughnose Sea Dragon; Figure 9 : Sample of Ge's sea bracelet fish.

[0029] Figure 10-18 The specificity map of the characteristic peptide SEQ ID NO.2 of the multispinous sea dragon provided by the present invention;

[0030] in: Figure 10 : SEQ ID NO.2 characteristic peptide control; Figure 11 : Sample of Multispined Sea Dragon; Figure 12 : Sample of Pseudohalodon; Figure 13 : Schlossosaurus sample; Figure 14 : Diao Hailong sample; Figure 15 : Sample of Baojia spear-nosed sea dragon; Figure 16 : Sample of Long Thick-snouted Sea Dragon; Figure 17 : Sample of Roughnose Sea Dragon; Figure 18 : Sample of Ge's sea bracelet fish. DETAILED DESCRIPTION

[0031] The present invention provides two characteristic polypeptides of the multi-spined sea dragon, and the amino acid sequences of the characteristic polypeptides are shown in SEQ ID NO.1: VGPAGPGGAAGPAGPGGPVGKDGAR, and SEQ ID NO.2: GA-(HYP)-GLGGPTGSR. The present invention provides peptides with characteristic identification significance for the multi-spined sea dragon. The present invention hydrolyzes the water extracts of different sea dragon species such as the multi-spined sea dragon, the sea dragon, the pseudo-sea dragon, and the schumeri sea dragon with trypsin, and then uses nano-liquid chromatography-high-resolution mass spectrometry to analyze the extracts to obtain a mass spectrum of the full peptide segment of the sea dragon protein; the mass spectrometry data is subjected to chemometric analysis, and the experimental results are specifically verified using triple quadrupole mass spectrometry; finally, two characteristic ion pairs are provided, corresponding to two characteristic peptide segments, and the specificity verification results are good.

[0032] The present invention also provides the use of the characteristic polypeptide described in the above technical solution in identifying the multispinous sea dragon.

[0033] In the present invention, the identification is performed using mass spectrometry, and the detected ion pairs include: a quantitative ion with a mass-to-charge ratio of m / z 676.3→418.2 for SEQ ID NO.1, and a qualitative ion with a mass-to-charge ratio of m / z 676.3→603.3; a quantitative ion with a mass-to-charge ratio of m / z 521.7→631.3 for SEQ ID NO.2, and a qualitative ion with a mass-to-charge ratio of m / z 521.7→517.2.

[0034] The present invention also provides a method for identifying the multispinous sea dragon based on the characteristic polypeptide described in the above technical solution, comprising the following steps:

[0035] (1) mixing the pretreated sea dragon extract or the pretreated sea dragon medicinal wine extract with trypsin to obtain a mixed solution, mixing the mixed solution with an aqueous ammonium bicarbonate solution, performing enzymatic hydrolysis and filtering, and taking the filtrate to obtain a test solution;

[0036] (2) Using the characteristic peptide of the multispinous sea dragon described in the above technical solution as a reference substance, adding ammonium bicarbonate aqueous solution to dissolve it to obtain a reference substance solution;

[0037] (3) Detection and analysis using triple quadrupole mass spectrometry;

[0038] There is no time sequence limitation between the steps (1) and (2).

[0039] The method described in the present invention extracts differential polypeptide segments from the protein sequence and uses mass spectrometry for detection. The operation is simple and fast, the measurement results are stable, and it is not affected by the morphology and processing of the sea dragon sample. It fills the gaps in the identification of the multi-spined sea dragon species and the detection method of the multi-spined sea dragon in sea dragon products, and provides a reference for the search for characteristic peptide segments in closely related species that are lacking in the database.

[0040] The present invention mixes the pretreated sea dragon extract or the pretreated sea dragon medicinal wine extract with trypsin to obtain a mixed solution, mixes the mixed solution with an aqueous ammonium bicarbonate solution, filters after enzymolysis, and takes the filtrate to obtain a test solution. In the present invention, the preparation method of the pretreated sea dragon extract preferably includes the following steps: crushing the sea dragon, mixing it with water and decocting it three times, the decoction time is 4h, 3h and 2h respectively, combining the decoctions, and diluting it with an aqueous ammonium bicarbonate solution to obtain a pretreated sea dragon extract. The present invention preferably takes 100g of crushed sea dragon, and the decoction is preferably 500mL in total. In the present invention, the dilution is preferably diluted 10 times. Specifically, the present invention preferably takes 5mL of the decoction, places it in a 50mL measuring flask, dilutes it to the scale with an aqueous ammonium bicarbonate solution, shakes well, and obtains a pretreated sea dragon extract. In the present invention, the preparation process of the pretreated sea dragon medicinal wine extract preferably includes the following steps: removing the sea dragon from the sea dragon medicinal wine, drying at low temperature, and decocting with water three times for 4 hours, 3 hours, and 2 hours, respectively. The decoctions are combined, filtered, and the filtrate is collected to obtain the pretreated sea dragon medicinal wine extract. After obtaining the pretreated sea dragon extract and the pretreated sea dragon medicinal wine extract, the present invention preferably measures 1-5 mL of the sea dragon extract or the sea dragon medicinal wine extract, preferably adds 100 μL of trypsin solution, shakes well, adds aqueous ammonium bicarbonate to 50 mL, shakes well, seals, and then performs enzymatic hydrolysis. In the present invention, the enzymatic hydrolysis conditions are preferably 37°C for 2 hours. After enzymatic hydrolysis, the present invention preferably cools, more preferably cools to room temperature, preferably ranging from 10 to 30°C. After cooling, the present invention filters, and the filtrate is used as the test solution.

[0041] In the present invention, 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 invention, the mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.

[0042] The present invention uses the characteristic peptide of the multi-spined sea dragon described in the above technical solution as a reference substance, dissolves it in water, and obtains a reference solution. The present invention does not specifically limit the source of the characteristic peptide of the multi-spined sea dragon, and can be synthesized using an artificial synthesis method. For example, the two characteristic peptides of the multi-spined sea dragon of the present invention were synthesized according to a specified amino acid sequence by Nanjing Source Peptide Biotechnology Co., Ltd.

[0043] The present invention utilizes triple quadrupole mass spectrometry for detection and analysis. In the present invention, the triple quadrupole mass spectrometry preferably uses 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and performs gradient elution: 0-3 min, 5% A→8% A; the flow rate is 0.5 ml per minute. In the present invention, the triple quadrupole mass spectrometry preferably uses a mass spectrometer detector, electrospray positive ion mode, and multiple reaction monitoring. SEQ ID NO.1 selects a quantitative ion with a mass-to-charge ratio of m / z 676.3→418.2 and a qualifier ion with a mass-to-charge ratio of m / z 676.3→603.3; SEQ ID NO.2 selects a quantitative ion with a mass-to-charge ratio of m / z 521.7→631.3 and a qualifier ion with m / z 521.7→517.2.

[0044] The following is a further detailed introduction to the two characteristic polypeptides of the multispinous sea dragon, their applications, and the method for identifying the multispinous sea dragon described in the present invention in conjunction with specific examples. The technical solutions of the present invention include but are not limited to the following examples.

[0045] Triple quadrupole mass spectrometry conditions

[0046] Gradient elution was performed (0-3 min, 5% A→8% A) using 0.1% formic acid in acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B; the flow rate was 0.5 ml / min. Multiple reaction monitoring (MRM) was performed using a mass spectrometer in positive electrospray ionization (ESI+) mode. For SEQ ID NO. 1, a quantification ion with a mass-to-charge ratio of m / z 676.3→418.2 and a qualifier ion with a mass-to-charge ratio of m / z 676.3→603.3 were selected; for SEQ ID NO. 2, a quantification ion with a mass-to-charge ratio of m / z 521.7→631.3 and a qualifier ion with a mass-to-charge ratio of m / z 521.7→517.2 were selected.

[0047] Example 1: Search for characteristic peptides of Diaothias multispinosa

[0048] (1) Preparation of test solution

[0049] Sea dragon water extract: Take 100g of the test sample sea dragon, grind it, place it in a conical flask, add water and boil it three times, for 4h, 3h, and 2h respectively. Combine the decoctions to make 500mL in total, boil gently and concentrate until the liquid becomes viscous, transfer it to a silica gel bowl, and dry it in a 60℃ electric constant temperature forced air drying oven until it becomes solid to obtain the sea dragon water extract;

[0050] Test solution: Weigh 0.1 g of the water extract of the sea dragon, add 50 ml of 1% ammonium bicarbonate aqueous solution, and sonicate for 30 min to dissolve it. Filter with a microporous membrane, take 100 μL of the filtrate, add 10 μL of 1 mg / ml trypsin aqueous solution, and enzymatically hydrolyze at 37°C for 2 h. Take out and cool to room temperature.

[0051] (2) Selection of characteristic ions and sequence inference

[0052] After the sample solution was analyzed by nano-liquid chromatography-high-resolution mass spectrometry, the mass spectrometry data was imported into PEAKS 8.5 software for de novo sequencing and sequence prediction of all peptides in the sample. The results were analyzed. From the analysis results, peptides that were only detected in D. multispinosa and almost not detected in other species of D. multispinosa were selected as parent ions. Their secondary spectra were analyzed one by one, and the daughter ions with the best response were selected. Further analysis revealed the mass spectrometric ion pairs representing the differences of D. multispinosa: m / z 676.3 (doubly charged) → 418.2, 603.3 and m / z 521.7 (doubly charged) → 631.3, 517.2. Further analysis revealed the characteristic peptide sequences corresponding to these ions: SEQ ID NO. 1: VGPAGPGGAAGPAGPGGPVGKDGAR and SEQ ID NO. 2: GA-(HYP)-GLGGPTGSR, where HYP represents hydroxyproline.

[0053] According to the inferred amino acid sequence, a characteristic peptide reference substance of the multi-spined sea dragon was commissioned to be synthesized. The reference substance and the multi-spined sea dragon test solution were tested simultaneously. The retention time and secondary mass spectrometry information of the two were consistent, thus confirming the correctness of the sequence.

[0054] Example 2: Study on the specificity of the characteristic peptide of Diaothias multispinosa

[0055] The water extracts of all species of sea dragons were tested using triple quadrupole mass spectrometry. The results showed that the three batches of sea dragons all had the characteristic peptide chromatographic peak of sea dragons, while the other sea dragons, ... Figure 1-9 , see attached Figure 10-18 The results showed that the characteristic peptide was unique to D. multispinata and could be used as an indicator component to identify D. multispinata.

[0056] Example 3: Application of the characteristic peptide of Pieris multispinosa in Pieris extract and Pieris medicinal wine

[0057] (1) Sample preparation

[0058] Preparation of the test solution of sea dragon extract: Take 100g of the test sea dragon, crush it, place it in a conical flask, add water and boil it three times, for 4h, 3h, and 2h respectively, and combine the decoctions to make 500mL in total; take 5ml of sea dragon extract, place it in a 50ml volumetric flask, add 1% ammonium bicarbonate aqueous solution to dilute to the scale, shake well, measure 1ml to a 5ml volumetric flask, add 100μl of 1mg / ml trypsin aqueous solution, shake well, and then add 1% ammonium bicarbonate aqueous solution to make up to the scale, shake well, seal it, and hydrolyze it at 37℃ for 2h. Cool it, filter it, and take the filtrate as the test solution.

[0059] Preparation of the test solution of Hailong medicinal wine: Take 10 g of Hailong from Hailong medicinal wine, dry it at low temperature, add water and boil it three times, for 4 h, 3 h, and 2 h respectively, combine the decoctions, a total of 50 mL; filter, take 1 ml of the filtrate and transfer it to a 5 ml volumetric flask, and prepare it according to the above method starting from "add 100 μl of 1 mg / ml trypsin aqueous solution".

[0060] Reference solution of the characteristic peptide fragment of the multi-spined sea dragon: weigh 10 mg each of the characteristic peptide reference substances SEQ ID NO.1 and SEQ ID NO.2 of the multi-spined sea dragon, place them in a 50 ml volumetric flask, add water to dissolve and dilute to the scale to obtain the solution.

[0061] (2) The determination was performed using triple quadrupole mass spectrometry conditions.

[0062] (3) Sample measurement

[0063] Ten batches of collected samples of Multispined Sea Dragon powder extract and eight batches of Multispined Sea Dragon medicinal wine were tested, and the results showed that the characteristic peptides of Multispined Sea Dragon were detected in all of them, indicating that both characteristic peptides can be used to identify Multispined Sea Dragon components in sea dragon products.

[0064] Example 4: Ion Information with Identification Potential

[0065] Five ions with identification potential were screened out from 368 candidate ions. After verification by triple quadrupole mass spectrometry, it was found that not all ions could be used to identify the components of Diao Hai Long. Some ions were not specific enough for Diao Hai Long. Only ions with mass-to-nuclear ratios of 676.3 and 521.7 had good specificity. The specific results are shown in Table 1.

[0066] Table 1 Specificity verification results

[0067]

[0068]

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A polypeptide characteristic of the multispinous sea dragon, characterized in that: The amino acid sequence of the characteristic polypeptide is shown in SEQ ID NO.1 and / or SEQ ID NO.

2.

2. Use of the characteristic polypeptide according to claim 1 in identifying the multispinous sea dragon.

3. The use according to claim 2, characterized in that The identification was performed using mass spectrometry, and the ion pairs detected for SEQ ID NO.1 included: a quantitative ion with a mass-to-charge ratio of m / z 676.3→418.2, and a qualitative ion with a mass-to-charge ratio of m / z 676.3→603.3; the ion pairs detected for SEQ ID NO.2 included: a quantitative ion with a mass-to-charge ratio of m / z 521.7→631.3, and a qualitative ion with a mass-to-charge ratio of m / z 521.7→517.

2.

4. A method for identifying the multispinous sea dragon, characterized in that: The following steps are involved: (1) Preparation of the test solution: the pretreated sea dragon extract or the pretreated sea dragon medicinal wine extract is mixed with trypsin to obtain a mixed solution, the mixed solution is mixed with an aqueous ammonium bicarbonate solution, and the mixture is filtered after enzymatic hydrolysis to obtain the test solution; (2) Preparation of reference solution: using the characteristic peptide of the multispinous sea dragon as the reference substance according to claim 1, dissolving it in water to obtain a reference solution; (3) Detection and analysis: Detection and analysis are performed using triple quadrupole mass spectrometry; There is no time sequence limitation between the steps (1) and (2); In step (1), the method for preparing the pretreated sea dragon extract comprises the following steps: crushing the sea dragon, mixing it with water, and decocting it three times, with the decocting time being 4 hours, 3 hours, and 2 hours respectively, combining the decoctions, and diluting them with an aqueous ammonium bicarbonate solution to obtain the pretreated sea dragon extract; The preparation process of the pretreated sea dragon medicinal wine extract comprises the following steps: taking out the sea dragon from the sea dragon medicinal wine, drying at low temperature, decocting with water three times, the decocting time being 4 hours, 3 hours and 2 hours respectively, combining the decoctions, filtering, and taking the filtrate to obtain the pretreated sea dragon medicinal wine extract; 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; The mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%; the enzymatic hydrolysis condition is 37° C. for 2 hours; The triple quadrupole mass spectrometry method used 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; the flow rate was 0.5 ml per minute; The triple quadrupole mass spectrometry method uses a mass spectrometer detector in electrospray positive ion mode to perform multiple reaction monitoring. For SEQ ID NO.1, a quantitative ion with a mass-to-charge ratio of m / z 676.3→418.2 and a qualitative ion with a mass-to-charge ratio of m / z 676.3→603.3 are selected as the detection ion pair. For SEQ ID NO.2, a quantitative ion with a mass-to-charge ratio of m / z 521.7→631.3 and a qualitative ion with a mass-to-charge ratio of m / z 521.7→517.2 are selected as the detection ion pair.

Images