Characteristic peptide fragment for identifying male and female ground beetles as well as detection method and application of characteristic peptide fragment

Abstract

The invention discloses a characteristic peptide fragment for identifying male and female ground beetles and a detection method of the characteristic peptide fragment, and the characteristic peptide fragment with the sequence of Glu-Met-Ser-Trp-Ile-Ala-Asp-Thr-Tyr-Ala-Lys, the sequence of Ile-Val-Pro-Ile-Val-Glu-Pro-Asp-Gly-Asp-His-Asp-Leu-Asp-Glu-Leu-Leu-Gly-Arg and the sequence of the characteristic peptide fragment with the sequence of Glu-Met-Ser-Trp-Ile-Asp-Thy-Lys, the sequence of the characteristic peptide fragment with the sequence of Ile-Val-Pro-Ile-Val-Glu The characteristic peptide fragments have high specificity and can be used for identifying drugs containing male ground beeltle medicinal materials. According to the method for detecting the characteristic peptide fragment of the male ground beeltle, the optimal enzymolysis condition, the chromatographic condition and the mass spectrum condition are screened out through a large number of experiments, the whole method is easy to operate and accurate in judgment, quality control over ground beeltle drugs is facilitated, and the method has important significance on quality control over the drugs containing the ground beeltle.

Description

Technical Field

[0001] This invention relates to a method for detecting and identifying animal drugs, specifically to a method for identifying characteristic peptides of male and female ground beetles, their detection methods, and their applications. Background Technology

[0002] Ground beetle, also known as earth beetle or ground cricket, is an insect belonging to the family Blattodea. Eupolyphaga sinensis Walker or Jidi Bie Steleophaga plancyi The dried female body of *Boleny*, a species first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), is listed as a medicinal material of medium medicinal value. Traditionally, it is highly regarded for its ability to promote blood circulation, eliminate blood stasis, remove thrombi, and promote tendon and bone healing. *Boleny* is now included in the 2025 edition of the *Chinese Pharmacopoeia*. It is used to break up blood stasis, promote tendon and bone healing, and is commonly used for traumatic injuries, fractures, amenorrhea due to blood stasis, postpartum abdominal pain due to blood stasis, and abdominal masses. Modern pharmacological research shows that *Boleny* has broad pharmacological effects in anti-tumor, analgesia, anticoagulation, and promoting fracture healing. Research on the material basis of *Boleny* indicates that its main chemical components are proteins, amino acids, polysaccharides, and volatile oils.

[0003] According to the Chinese Pharmacopoeia, only female *Eupolyphaga sinensis* (a type of ground beetle) are approved for medicinal use. However, confusion between male and female *Eupolyphaga sinensis* exists in the market, and traditional methods are insufficient to distinguish between them. This invention identifies three characteristic peptide markers of male *Eupolyphaga sinensis* through experimental screening. These markers can be used to detect the presence of males in female *Eupolyphaga sinensis*, and can also detect the adulteration ratio of males in different *Eupolyphaga sinensis* samples. This research provides a practical molecular tool for the quality control and standardization of *Eupolyphaga sinensis* medicinal materials. Summary of the Invention

[0004] The main objective of this invention is to address the aforementioned problems and shortcomings by providing a characteristic peptide for identifying male Eupolyphaga sinensis and a method for its detection. The Eupolyphaga sinensis characteristic peptide provided by this invention exhibits high specificity. The provided method is simple to operate, highly sensitive, and can accurately identify whether female Eupolyphaga sinensis medicinal materials contain male Eupolyphaga sinensis components, providing a scientific method for ensuring the quality of Eupolyphaga sinensis and its products.

[0005] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows: Characteristic peptides used to identify male and female ground beetles include any one of the following P1 to P3 or a combination thereof. P1: Glu-Met-Ser-Trp-Ile-Ala-Asp-Thr-Tyr-Ala-Lys P2: Ile-Val-Pro-Ile-Val-Glu-Pro-Glu-Val-Leu-Pro-Asp-Gly-Asp-His-Asp-Leu-Asp-Arg P3: Thr-Gly-Ala-Ile-Val-Asp-Val-Pro-Val-Gly-Asp-Glu-Leu-Leu-Gly-Arg.

[0006] Reference peptide PR: Glu-Ala-Phe-Ser-Leu-Phe-Asp-Lys-Asp-Gly-Asp-Gly-Thr-Ile-Thr-Thr-Lys.

[0007] The above-mentioned P1~P3 and reference peptide PR, converted to amino acid abbreviations, are as follows: P1: EMSWIADTYAK P2: IVPIVEPEVLPDGDHDLDR P3: TGAIVDVPVGDELLGR PR: EAFSLFDKDGDGTITTK The reference peptide PR used in this invention is present in both male and female Eupolyphaga sinensis, and the content is similar. This invention uses this reference peptide to homogenize the relative content of characteristic peptides in male and female Eupolyphaga sinensis, which can more accurately detect the content of characteristic peptide segments P1~P3 in male and female Eupolyphaga sinensis.

[0008] A method for detecting characteristic peptides of Eupolyphaga sinensis, comprising the following steps: (1) Prepare a reference solution by mixing the characteristic peptides P1~P3 used to identify ground beetle and the reference peptide PR. (2) After digesting the *Eupolyphaga sinensis* animal drug sample with trypsin, the enzymatic digest and the mixed control solution of characteristic peptides used to identify *Eupolyphaga sinensis* in step (1) were injected into a liquid chromatography-mass spectrometry (LC-MS) instrument. Characteristic peptides P1~P3 and reference peptide PR were used as controls. Multiple reaction monitoring (MRM) was employed, and the ion pairs selected were: P1: 657.9→781.4 (for relative content detection), 657.9→668.2; P2: 710.4→639.5 (for relative content detection), 710.4→739.6; P3: 806.1→955.2 (for relative content detection), 806.1→343.1; PR: 615.6→649.5, 615.6→823.1 (for relative content detection); The test is performed; if the above ion pairs are detected, and the retention time of the ions is consistent with the characteristic peptide reference standard used to identify *Eupolyphaga sinensis*, and the daughter ions are consistent with the daughter ions of the reference standard, wherein the relative contents of the characteristic peptide P1 ion pair (657.9 > 781.4), P2 ion pair (710.4 > 639.5), and P3 ion pair (806.1 > 955.2) in male *Eupolyphaga sinensis* and the reference peptide ion pair PR (615.6 > 823.1) are 10.0, 537.3, and 126.2 or higher, respectively, then the sample to be tested contains male *Eupolyphaga sinensis* components.

[0009] If the relative contents of the characteristic peptide ion pair P1 (657.9 > 781.4), P2 (710.4 > 639.5), and P3 (806.1 > 955.2) in the female ground beetle are less than 1.7, 294.2, and 50.3 respectively, and are the reference peptide ion pair (615.6 > 823.1), then the sample to be tested contains female ground beetle components.

[0010] As a preferred embodiment, the detection method for identifying characteristic peptides of Eupolyphaga sinensis described above includes the following enzymatic digestion method: Take the male and female Eupolyphaga sinensis animal drug samples to be tested, add Tris-HCl buffer to each to reconstitute, sonicate until completely dissolved, take 1600 μg of protein, add Tris-HCl buffer to dilute, add trypsin, and incubate in a constant temperature water bath to obtain the final product.

[0011] As a preferred embodiment, the detection method for identifying characteristic peptides of Eupolyphaga sinensis described above includes the following enzymatic digestion method: Take 30 mg of each male and female Eupolyphaga sinensis animal drug sample to be tested, add 1 mL of pre-cooled 4% SDS solution, grind into a homogenate, centrifuge and collect the supernatant, determine the protein content using BCA, take 1600 μg of protein, add 50 mM Tris-HCl buffer (pH 8.8) and dilute to 200 μl, incubate with DTT and IAA, precipitate with 80% acetone at −20 °C for 4 h, centrifuge and discard the supernatant, evaporate the acetone, reconstitute with 8M urea, and dilute to 1M with 50 mM Tris-HCl buffer (pH 8.8). Add 40 μl of 0.1 μg / μl trypsin, incubate in a constant temperature water bath at 37 °C for 12 h, desalt using a Seppak C18 desalting column, evaporate to dryness, and reconstitute with 10% acetonitrile to obtain the final product.

[0012] As a preferred embodiment, the detection method for identifying characteristic peptides of Eupolyphaga sinensis described above uses the following liquid chromatography-mass spectrometry (LC-MS) conditions: 1.7 μm Acquity UPLC C20 column. 18The column was 2.1 μm × 100 mm, the sample loading volume was 2 μl, the flow rate was 0.3 ml / min, the mobile phase A was acetonitrile containing 0.1% formic acid, and the mobile phase B was water containing 0.1% formic acid. The elution mode was: 0–2 min, 10–30% A linear gradient elution; 2–6 min, 30–50% A linear gradient elution; 6–7 min, 50% A isocratic elution. Triple quadrupole mass spectrometry was used, and the mass spectrometry parameters were: ion source temperature 500 ℃; ionization voltage 5500 V; desolventizing temperature 500 ℃; ion source gas 1, 60 psi; ion source gas 2, 60 psi.

[0013] The ion pairs in MRM mode are: .

[0014] Beneficial effects: Through extensive experimental screening, this invention has identified three characteristic peptides that can be used to identify male Eupolyphaga sinensis. These three characteristic peptides for identifying male Eupolyphaga sinensis have high specificity and can be used to identify medicines containing male Eupolyphaga sinensis. The characteristic peptides provided by this invention can be used as markers for identifying male Eupolyphaga sinensis.

[0015] The present invention provides a method for detecting characteristic peptides of *Eupolyphaga sinensis*, which, through extensive experimental screening, has optimized enzymatic hydrolysis, chromatographic, and mass spectrometric conditions. The entire method is simple to operate, accurate, and can precisely distinguish characteristic peptides used to identify male *Eupolyphaga sinensis*. The characteristic peptides and detection method for identifying *Eupolyphaga sinensis* provided by this invention are beneficial for the quality control of *Eupolyphaga sinensis*-containing pharmaceuticals and are of great significance for ensuring the quality of pharmaceuticals containing *Eupolyphaga sinensis*. Attached Figure Description

[0016] Figure 1 The flowchart shows the screening process for characteristic peptides P1, P2, P3 and internal reference peptide PR.

[0017] Figure 2 Mass spectra of characteristic peptides P1, P2, P3 and internal reference peptide PR.

[0018] Figure 3 XIC chromatogram of a reference standard for identifying characteristic peptide fragments of Eupolyphaga sinensis.

[0019] Figure 4 XIC plot of a sample used to distinguish between characteristic peptides from male and female Eupolyphaga sinensis samples.

[0020] Figure 5 XIC plot of characteristic peptide samples used to identify different ratios of male and female ground beetles. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0022] Example 1: Screening of characteristic peptides P1, P2, P3 and internal reference peptide PR Take 30 mg of male Eupolyphaga sinensis animal drug sample, add 1 mL of pre-cooled 4% SDS solution, grind into a homogenate, centrifuge and collect the supernatant. Determine protein content using BCA. Take 1600 μg of protein, add 50 mM Tris-HCl buffer (pH 8.8) and dilute to 200 μl. Incubate with DTT and IAA, precipitate with 80% acetone at −20 °C for 4 h, centrifuge, discard the supernatant, evaporate acetone, reconstitute with 8M urea, and dilute to 1M with 50 mM Tris-HCl buffer (pH 8.8). Add 40 μl of 0.1 μg / μl trypsin, incubate at 37 °C for 12 h, Seppak C... 18 The sample was desalted using a desalting column, evaporated to dryness, and then reconstituted with 10% acetonitrile. This method yielded a mixed peptide sample of male Eupolyphaga sinensis (male ES).

[0023] Take 30 mg of female ground beetle animal drug sample and prepare female ground beetle mixed peptide sample (female ES) using the same method.

[0024] like Figure 1 35 μl of male and female ES samples were taken and diluted to 100 μl with 0.5 M triethylamine bicarbonate (TEAB) buffer. Peptides derived from female ES were labeled with a light reagent (formaldehyde HCHO), while peptides derived from male ES were labeled with a medium reagent (deuterated formaldehyde DCDO). Then, 0.6 M NaBH3CN was added to the samples on ice to label the N-terminus and amino group (NH2) of lysine with isotopic tags. After labeling, equal volumes of light-labeled and medium-labeled samples were mixed, desalted using a Waters C18 Sep-Pak column, concentrated by centrifugation, dried, reconstituted with the initial mobile phase, and analyzed by nanoLC-MS / MS.

[0025] The obtained MS / MS data were analyzed using PEAKS Studio software, with a search of the UniProt insect database. Trypsin was selected as the digestive enzyme, allowing a maximum of two cleavage deletions. Fixed modifications were set as follows: ureidomethylation (+57.02 Da), deamidation (+0.98 Da), oxidation (+15.99 Da), hydroxylation (+15.99 Da), light labeling at the lysine or N-terminus (+28.03 Da), and medium labeling (+32.06 Da) were set as variable modifications. All other parameters were set to default, with a fragment ion tolerance of 0.03 Da and a maximum precursor ion tolerance of 25 ppm. Dimethyl labeling quantification was performed using the PEAKS Q module in PEAKS Studio 8.5 for relative quantification of lightly and medium-labeled peptides at the mass spectrometry level, with a mass error tolerance of 15.0 ppm and a retention time range of 2.0 minutes.

[0026] A total of 277 peptides were identified. In order to screen potential peptide biomarkers of male Eupolyphaga sinensis, screening criteria and thresholds were set: (1) the peptide must have trypsin digestion characteristics; (2) the fold change (i.e. male / female) > 8 and the p value < 0.01; (3) identification confidence: −10lgP value ≥ 50 and have high-quality MS / MS spectra. This screening identified three peptide biomarkers: P1 (Glu-Met-Ser-Trp-Ile-Ala-Asp-Thr-Tyr-Ala-Lys), P2 (Ile-Val-Pro-Ile-Val-Glu-Pro-Glu-Val-Leu-Pro-Asp-Gly-Asp-His-Asp-Leu-Asp-Arg), and P3 (Thr-Gly-Ala-Ile-Val-Asp-Val-Pro-Val-Gly-Asp-Glu-Leu-Leu-Gly-Arg). Mass spectra are shown below. Figure 2 .

[0027] In addition, to standardize the relative abundance of peptides, this invention screened for internal control peptides. Conditions (1) and (3) remained unchanged, while condition (2) was set to a fold change between 0.95 and 1.05 with a p-value > 0.05. Through this screening process, an internal control peptide PR (Glu-Ala-Phe-Ser-Leu-Phe-Asp-Lys-Asp-Gly-Asp-Gly-Thr-Ile-Thr-Thr-Lys) was selected.

[0028] Example 2

[0029] A characteristic peptide for identifying the sex of male ground beetles, wherein the sequences of characteristic peptides 1-3 are shown in Sequence Listings 1-3, and the sequence of the reference peptide is shown in Sequence Listing 4.

[0030] The characteristic peptide segments are: P1: Glu-Met-Ser-Trp-Ile-Ala-Asp-Thr-Tyr-Ala-Lys P2: Ile-Val-Pro-Ile-Val-Glu-Pro-Glu-Val-Leu-Pro-Asp-Gly-Asp-His-Asp-Leu-Asp-Arg P3: Thr-Gly-Ala-Ile-Val-Asp-Val-Pro-Val-Gly-Asp-Glu-Leu-Leu-Gly-Arg Reference peptide PR: Glu-Ala-Phe-Ser-Leu-Phe-Asp-Lys-Asp-Gly-Asp-Gly-Thr-Ile-Thr-Thr-Lys.

[0031] (1) Take 30 mg of each of the female and male Eupolyphaga sinensis samples (n = 7) to be tested, add 1 mL of pre-cooled 4% SDS solution, grind and homogenize, centrifuge and collect the supernatant, determine the protein content by BCA, take 1600 μg of protein, add 50 mM Tris-HCl buffer at pH 8.8 and dilute to 200 μl, incubate with DTT and IAA, precipitate with 80% acetone at −20 ℃ for 4 h, centrifuge and discard the supernatant, evaporate the acetone, reconstitute with 8M urea and dilute to 1M with 50 mM Tris-HCl buffer at pH 8.8. Add 40 μl of 0.1 μg / μl trypsin respectively, incubate in a constant temperature water bath at 37 ℃ for 12 h, Seppak C 18 The salt is removed by desalting column, evaporated to dryness, and then reconstituted with 10% acetonitrile to obtain the final product.

[0032] (2) Prepare 1 ml of characteristic peptide reference solution with a concentration of 100 ng / ml for characteristic peptides P1~P3 and reference peptide PR.

[0033] (3) The enzymatic hydrolysate of male and female ground beetles from step (1) and the characteristic peptides P1~P3 and reference peptide PR from step (2) were detected by liquid chromatography-mass spectrometry (LC-MS). The LC conditions were as follows: the column was a 1.7 μm Acquity UPLC C18 column with a size of 2.1 μm × 100 mm, the sample loading volume was 2 μl, the flow rate was 0.3 ml / min, the mobile phase A was acetonitrile containing 0.1% formic acid, the mobile phase B was water containing 0.1% formic acid, and the elution method was as follows: 0~2 min, 10~30% A linear gradient elution, 2~6 min, 30~50% A linear gradient elution, 6~7 min, 50% A isocratic elution. Triple quadrupole mass spectrometry was used, and the mass spectrometry parameters were as follows: ion source temperature 500 ℃; ionization voltage 5500 V; desolventizing temperature 500 ℃; ion source gas 1, 60 psi; ion source gas 2, 60 psi. The ion pairs in MRM mode are shown in Table 1 below; Table 1

[0034] Table 2. Relative peak areas of characteristic peptides P1, P2, and P3 (P1~3 / PR)

[0035] See results Figure 3 , Figure 4 As shown in Table 2, ion peaks consistent with the characteristic peptide reference solution were detected in male *Eupolyphaga sinensis* samples, while very low levels were found in female samples. To maximize the differentiation of the relative peak areas of characteristic peptides between male and female insects, we used ion pairs with high response values ​​for characteristic peptides P1-P3 and ion pairs with low response values ​​for the reference peptide PR. The relative contents of characteristic peptides P1 (657.9 > 781.4), P2 (710.4 > 639.5), and P3 (806.1 > 955.2) in male *Eupolyphaga sinensis*, and the relative contents of the reference peptide PR (615.6 > 823.1) were 12.54, 671.57, and 157.75, respectively.

[0036] The relative contents of the characteristic peptide ion pairs P1 (657.9 > 781.4), P2 (710.4 > 639.5), and P3 (806.1 > 955.2) in female ground beetles, and the reference peptide ion pair PR (615.6 > 823.1) were less than 1.45, 245.15, and 41.9, respectively.

[0037] By lowering the minimum relative content of characteristic peptides in male insects by 20% and raising the maximum relative content in female insects by 20%, the relative contents of P1, P2, P3 and the reference peptide ion to PR in male insects are specified to be above 10.0, 537.3, and 126.2, respectively; while the relative contents of P1, P2, P3 and the reference peptide ion to PR in female insects are below 1.7, 294.2, and 50.3, respectively. Example 2 illustrates that the present invention can specifically detect characteristic peptide components in male *Eupolyphaga sinensis*, thereby distinguishing them from female *Eupolyphaga sinensis*.

[0038] Example 3: Detection of characteristic polypeptides in male and female ground beetles at different ratios (1) Take the female and male powders to be tested, and add male ground beetles to the female ground beetles at proportions of 0%, 20%, 40%, 60%, 80%, and 100%, for a total of 6 portions, each containing 30 mg, and mix well. Add 1 mL of pre-cooled 4% SDS solution, grind and homogenize, centrifuge and collect the supernatant, determine the protein content by BCA, take 1600 μg of protein, add 50 mM Tris-HCl buffer at pH 8.8 and dilute to 200 μl, incubate with DTT and IAA, precipitate with 80% acetone at −20 ℃ for 4 h, centrifuge and discard the supernatant, evaporate the acetone, reconstitute with 8M urea and dilute to 1M with 50 mM Tris-HCl buffer at pH 8.8. Add 40 μl of 0.1 μg / μl trypsin, incubate in a 37 ℃ water bath for 12 h, desalt using a Seppak C18 desalting column, evaporate to dryness, and reconstitute with 10% acetonitrile to obtain the final product.

[0039] (2) Prepare 1 ml of characteristic peptide reference solution with a concentration of 100 ng / ml by preparing characteristic peptide P1~P3 and reference peptide PR as described in Example 2.

[0040] (3) The enzymatic hydrolysate of male and female ground beetles from step (1) and the reference solution of characteristic peptides from step (2) were detected by liquid chromatography-mass spectrometry (LC-MS). The LC conditions were as follows: the column was a 1.7 μm Acquity UPLC C18 column with a specification of 2.1 μm × 100 mm, the sample loading volume was 2 μl, the flow rate was 0.3 ml / min, the mobile phase A was acetonitrile containing 0.1% formic acid, the mobile phase B was water containing 0.1% formic acid, and the elution method was as follows: 0~2 min, 10~30% A linear gradient elution, 2~6 min, 30~50% A linear gradient elution, 6~7 min, 50% A isocratic elution. Triple quadrupole mass spectrometry was used, and the mass spectrometry parameters were as follows: ion source temperature 500 ℃; ionization voltage 5500V; desolventizing temperature 500 ℃; ion source gas 1, 60 psi; ion source gas 2, 60 psi. The ion pairs in the MRM mode are the same as those in Table 1.

[0041] See results Figure 5In a mixture of male and female Eupolyphaga sinensis, the area of ​​the characteristic peptide peak showed a positive linear relationship with the increase of the proportion of male Eupolyphaga sinensis. This indicates that the method can also be used to detect whether female Eupolyphaga sinensis powder is adulterated with male Eupolyphaga sinensis, and the specific proportion of adulteration can be determined based on the linear relationship. Therefore, controlling the quality of female Eupolyphaga sinensis is of great significance for ensuring clinical efficacy.

[0042] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A characteristic peptide segment used to distinguish between male and female ground beetles, characterized in that, The characteristic peptide segments include any one of the following P1 to P3 or a combination thereof; P1: Glu-Met-Ser-Trp-Ile-Ala-Asp-Thr-Tyr-Ala-Lys P2: Ile-Val-Pro-Ile-Val-Glu-Pro-Glu-Val-Leu-Pro-Asp-Gly-Asp-His-Asp-Leu-Asp-Arg P3: Thr-Gly-Ala-Ile-Val-Asp-Val-Pro-Val-Gly-Asp-Glu-Leu-Leu-Gly-Arg Reference peptide PR: Glu-Ala-Phe-Ser-Leu-Phe-Asp-Lys-Asp-Gly-Asp-Gly-Thr-Ile-Thr-Thr-Lys.

2. A method for detecting characteristic peptides used to distinguish between male and female ground beetles, characterized in that, Includes the following steps: (1) Prepare a reference solution by preparing the characteristic peptides P1~P3 and the reference peptide PR according to claim 1; (2) After digesting the male and female Eupolyphaga sinensis samples with trypsin, the digest and the characteristic peptide control solution from step (1) were injected into the liquid chromatography-mass spectrometry (LC-MS) instrument. The characteristic peptides P1~P3 and the reference peptide PR were used as controls. The multiple reaction monitoring (MRM) mode was adopted, and the selected ion pairs were: P1：657.9→781.4、657.9→668.2； P2：710.4→639.5、710.4→739.6； P3：806.1→955.2、806.1→343.1； PR: 615.6→649.5, 615.6→823.1; If the above ion pairs are detected, and the retention time of the ions is consistent with that of the characteristic peptide reference standard, and the daughter ion is consistent with that of the reference standard, and the relative content of the male P1~P3 characteristic peptide ion pairs and the reference peptide PR ion pairs is higher than that of the female, then the detection of these ion pairs is also important.

3. The method for detecting characteristic peptides for identifying male and female Eupolyphaga sinensis according to claim 2, characterized in that, The enzyme digestion method is as follows: Take the male and female *Eupolyphaga sinensis* animal drug samples to be tested, add Tris-HCl buffer to each to reconstitute, sonicate until completely dissolved, take 1600 μg of protein, add Tris-HCl buffer to dilute, add trypsin, and incubate in a constant temperature water bath to obtain the protein.

4. The detection method for identifying characteristic peptides of Eupolyphaga sinensis according to claim 3, characterized in that, The enzyme digestion method is as follows: Take 30 mg each of the male and female *Eupolyphaga sinensis* animal drug samples to be tested, add 1 mL of pre-cooled 4% SDS solution, grind and homogenize, centrifuge and collect the supernatant, determine the protein content by BCA, take 1600 μg of protein, add 50 mM Tris-HCl buffer (pH 8.8) and dilute to 200 μl, incubate with DTT and IAA, precipitate with 80% acetone at −20 °C for 4 h, centrifuge and discard the supernatant, evaporate the acetone, reconstitute with 8M urea, and dilute to 1M with 50 mM Tris-HCl buffer (pH 8.8). Add 40 μl of 0.1 μg / μl trypsin and incubate at 37 ℃ for 12 h. Seppak C 18 The salt is removed by desalting column, evaporated to dryness, and then reconstituted with 10% acetonitrile to obtain the final product.

5. The method for detecting characteristic peptides for identifying male Eupolyphaga sinensis according to claim 2, characterized in that, The liquid chromatography conditions for LC-MS were as follows: 1.7 μm column, Acquity UPLC C 18 The column was 2.1 μm × 100 mm, the sample loading volume was 2 μl, and the flow rate was 0.3 ml / min. Mobile phase A was acetonitrile containing 0.1% formic acid, and mobile phase B was water containing 0.1% formic acid. The elution mode was: 0–2 min, 10–30% A linear gradient elution; 2–6 min, 30–50% A linear gradient elution; 6–7 min, 50% A isocratic elution. Triple quadrupole mass spectrometry was used, with the following parameters: ion source temperature 500 ℃; ionization voltage 5500 V; desolventizing temperature 500 ℃; ion source gas 1, 60 psi; ion source gas 2, 60 psi. The ion pairs in MRM mode are: 。 6. The use of the characteristic peptide segment of claim 1 in the identification of male and female ground beetles.

7. The application of the characteristic peptide segment of claim 1 in the identification of male Eupolyphaga sinensis markers.

8. The application according to claim 6 or 7, characterized in that, The relative contents of the characteristic peptide ion pairs P1 (657.9 > 781.4), P2 (710.4 > 639.5), and P3 (806.1 > 955.2) in male Eupolyphaga sinensis, and the reference peptide ion pair PR (615.6 > 823.1) were 10.0, 537.3, and 126.2 or higher, respectively.

9. The application according to claim 6 or 7, characterized in that, The relative contents of the characteristic peptide ion pairs P1 (657.9 > 781.4), P2 (710.4 > 639.5), and P3 (806.1 > 955.2) in female Eupolyphaga sinensis, and the reference peptide ion pair PR (615.6 > 823.1) were 1.7, 294.2, and 50.3 or less, respectively.

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