Lumbricus marker polypeptide and application thereof in earthworm identification

Abstract

The invention discloses an earthworm marker polypeptide and application thereof in earthworm identification. The invention provides a method for identifying a rare variety of Huniang pheretima, namely chlamys farreri, and whether a sample to be detected contains the chlamys farreri or not is judged by analyzing whether the sample to be detected contains polypeptide as shown in SEQ ID NO.4 (PP6-3) or not by adopting a pheretima liquid chromatography-mass spectrometry identification method. The invention also provides a characteristic polypeptide of pheretima chlamys. The polypeptide has the advantage of good specificity.

Description

Technical Field

[0001] The present application relates to the field of analysis and detection, and in particular to a characteristic peptide of earthworm and its application in earthworm identification. Background Art

[0002] The original animal of earthworm is earthworm. The 2020 edition of the Chinese Pharmacopoeia records that the source of earthworm is the dried body of Pheretima aspergillum (E.Perrier) (abbreviated as PA), Pheretima vulgaris Chen (PV), Pheretima guillelmi (Michaelsen) (PG) or Pheretima nectarinifera Michaelsen (PP) of the family Corymbidae. The former is commonly known as "Guangdilong" and the latter three are commonly known as "Shanghaidilong". Earthworm has the traditional effects of clearing away heat and calming convulsions, unblocking meridians, relieving asthma and diuresis, and is widely used in clinical practice. The 2020 edition of the Chinese Pharmacopoeia uses thin layer chromatography to identify lysine, leucine, valine or compares with control medicinal materials, and the quality control indicators lack specificity. At present, there are 50 prescriptions containing earthworms in the pharmacopoeia, but there is a general lack of quality control items related to earthworms.

[0003] In recent years, medicinal earthworm resources have been in short supply, prices have gradually risen, and samples processed from non-medicinal earthworms have been mixed into the earthworm medicinal material market. According to literature reports, 22% of the medicinal material market is Pheretima annuli (i.e. Guangdong earthworm), 22% is Pheretima vulgaris (a type of Shanghai earthworm), Pheretima william and Pheretima fasciatus are rare (the other two types of Shanghai earthworms), and 55% is non-pharmacopoeia-based species, mainly Metaphire magna (abbreviated MM, similar in properties to Guangdong earthworm, mainly produced in Hainan), which shows that the earthworm varieties on the market are confused.

[0004] Establishing an exclusive quality control method with characteristic peptides as indicators can effectively realize the identification of earthworms and the inspection of common counterfeits and adulterated products. The prior art discloses the identification of Guangdong earthworms and a type of Shanghai earthworms (common earthworms) as well as the differentiation of counterfeits of Baoning earthworms. In previous studies, the inventors collected four authentic earthworms and common counterfeits, carried out non-labeled quantitative proteomics research, and screened the characteristic peptides of Guangdong earthworms, Shanghai earthworms and Baoning earthworms through multivariate statistical analysis, and established the identification of earthworms and the inspection of Baoning earthworms. Among them, the identification of Shanghai earthworms is aimed at common earthworms and William earthworms. The characteristic peptides of Ctenopharynx blindus were found to have poor specificity in later verification, so it is necessary to continue screening the characteristic peptides of Ctenopharynx blindus.

[0005] The traditional characteristic peptide screening strategy is mainly based on differential analysis of peptide identification results to determine species-specific peptides. This method relies on the proteome database of the research species and the number of peptide identifications. However, due to the general lack of proteome databases for the original animal species of traditional Chinese medicine, the amount of identified peptide data is small and the accuracy is poor, making it difficult to screen out characteristic peptides. The inventors found in the study that the characteristic peptides found in the traditional analysis strategy are poorly specific because the protein database used is not derived from Pheretima sphenanthera, and cannot be used for quality control research. Summary of the invention

[0006] The purpose of the present application is to provide a method for identifying Pheretima sphenanthera.

[0007] Another object of the present application is to provide a characteristic polypeptide for identifying Pheretima pubescens.

[0008] Another object of the present application is to provide a method for screening characteristic polypeptides of earthworms.

[0009] In order to solve the above technical problems, the first aspect of the present application provides a method for identifying Pheretima sphenanthera, the method comprising the steps of:

[0010] (1) Providing a sample of earthworm to be tested;

[0011] (2) Detecting the content of the polypeptide shown in SEQ ID NO.4 (PP6-3) in the earthworm sample, and determining whether the earthworm sample contains the polypeptide shown in SEQ ID NO.4 (PP6-3) by analyzing whether the earthworm sample contains the polypeptide shown in SEQ ID NO.4 (PP6-3).

[0012] In some preferred embodiments, the earthworm sample is medicinal earthworm.

[0013] In some preferred embodiments, in step (2), the sample to be tested is detected using a liquid chromatography tandem triple quadrupole mass spectrometer, and the mass spectrometry conditions of the liquid chromatography tandem triple quadrupole mass spectrometer include:

[0014] m / z 701.3 (doubly charged)→968.5 was set as the quantitative ion, and m / z 701.3 (doubly charged)→881.4 was set as the qualifier ion for detection.

[0015] In some preferred embodiments, the mass spectrometry conditions of the liquid chromatography tandem triple quadrupole mass spectrometer further include:

[0016] Electrospray positive ionization mode (ESI + ),

[0017] Detection mode: Multiple reaction monitoring (MRM).

[0018] In some preferred embodiments, the mass spectrometry conditions of the liquid chromatography tandem triple quadrupole mass spectrometer further include:

[0019] Dryer temperature 180-220°C (e.g. 200°C);

[0020] Gas flow rate 10-15 L / min (e.g. 13.0 L / min);

[0021] Atomizer 30-40 psi (e.g. 35 psi);

[0022] Sheath gas flow rate 10-15 L / min (e.g. 12 L / min);

[0023] The fragmentation voltage is 350-400V (eg 380V).

[0024] In some preferred embodiments, the chromatographic conditions of the liquid chromatography tandem triple quadrupole mass spectrometer include:

[0025] Acetonitrile was used as mobile phase A and 0.1% formic acid solution was used as mobile phase B for gradient elution.

[0026] In some preferred embodiments, the chromatographic conditions of the liquid chromatography tandem triple quadrupole mass spectrometer include:

[0027] The procedure of the gradient elution is: within 0 to 15 minutes, the volume proportion of mobile phase A is increased from 13% to 32% at a constant speed, and the volume proportion of mobile phase B is decreased from 87% to 68% at a constant speed.

[0028] In some preferred embodiments, the chromatographic conditions of the liquid chromatography tandem triple quadrupole mass spectrometer further include:

[0029] Use a C18 column (e.g., CORTECS T3 C18 column (2.7 μm, 2.1 × 100 mm);

[0030] Injection volume: 5 μL;

[0031] Flow rate: 0.4 mL / min.

[0032] In some preferred embodiments, the liquid chromatography-mass spectrometer is an Agilent 1290 ultra high performance liquid chromatograph-Agilent 6495 triple quadrupole mass spectrometer.

[0033] In a second aspect of the present application, a characteristic polypeptide for identifying Pheretima sphenanthera is provided, wherein the amino acid sequence of the characteristic polypeptide is selected from at least one of SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.5.

[0034] In a preferred embodiment, the amino acid sequence of the characteristic polypeptide is shown as SEQ ID NO.4.

[0035] The third aspect of the present application provides the characteristic polypeptide as described in the second aspect of the present invention, which is used in identifying earthworm samples.

[0036] In a preferred embodiment, the characteristic polypeptide is used as a standard for identifying earthworm samples.

[0037] In a preferred embodiment, the earthworm sample is a medicinal earthworm.

[0038] The fourth aspect of the present application provides a method for screening a characteristic polypeptide of earthworms, the method comprising the steps of:

[0039] S1, obtaining an original data table of earthworm samples; comprising the steps of: using a high-resolution mass spectrometer to analyze authentic earthworms, counterfeit earthworms and quality control samples, extracting ion information to obtain an original data table of earthworm samples containing retention time, mass-to-charge ratio, and normalized peak intensity information;

[0040] S2, remove the non-compliant data in the original data table, and use PCA analysis to analyze the original data to screen the target earthworm sample peaks.

[0041] S3, obtaining the differential ions of the target earthworm sample by comparing the difference between the target earthworm sample peak and other peaks;

[0042] S4, screening differentiated ions with strong specificity by performing specificity analysis on the differentiated ions;

[0043] S5, by De novo sequencing, the peptide sequences of the highly specific differential ions described in S4 are resolved;

[0044] S6, verify the accuracy of the polypeptide sequence.

[0045] In some preferred embodiments, the verification of the accuracy of the polypeptide sequence includes: comparing the retention time of the reference substance, the sample and the added sample, and comparing the mass-to-charge ratio of the parent ion and the daughter ion of the reference substance, the sample and the added sample.

[0046] In some preferred embodiments, orthogonal partial least squares discriminant analysis or variance analysis is used to obtain differentiated ions of the target earthworm sample in S3.

[0047] In some preferred embodiments, in S4, the daughter ions of the differential ions are screened by secondary mass spectrometry analysis, and then the differential ion specificity is verified by liquid chromatography tandem triple quadrupole mass spectrometry.

[0048] In some preferred embodiments, the characteristic polypeptide of earthworms is a characteristic polypeptide of Pheretima sphenanthera, and the method comprises the steps of:

[0049] S1, obtaining the original data table of earthworm samples from various sources; comprising the steps of: using high-resolution mass spectrometry to analyze various authentic earthworms, counterfeit earthworms and quality control samples, extracting ion information to obtain the original data table of earthworm samples containing retention time, mass-to-charge ratio, and normalized peak intensity information;

[0050] S2, remove the non-compliant data in the original data table, and use PCA analysis to analyze the original data to screen the peaks of Pheretima sphenanthera samples;

[0051] S3, using Pheretima sphenanthera as the first group and L. humilis and its other fakes as the second group for t-test analysis, several differential screening ions were obtained;

[0052] S4, screening the Pheretima sphenanthera differentiated ions with strong specificity by performing specificity analysis on the plurality of Pheretima sphenanthera differentiated ions;

[0053] S5, by De novo sequencing, the peptide sequence of the highly specific differential ions of Pheretima sphenanthera described in S4 was analyzed;

[0054] S6, verifying the accuracy of the polypeptide sequence, that is, obtaining a polypeptide characteristic of Pheretima pubescens.

[0055] Compared with the prior art, the present invention has at least the following advantages:

[0056] The present invention has developed a novel polypeptide screening method. Compared with the existing method, the present invention screened out the characteristic polypeptide of another rare species of Shanghai earthworm, Pheretima sphenanthera, which has the advantage of good specificity. The present invention also established an identification method and improved the liquid chromatography-mass spectrometry identification method of earthworm.

[0057] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0059] Figure 1 It is a PCA analysis diagram according to the embodiment of the present application;

[0060] Figure 2 It is a high-resolution secondary mass spectrum of representative ions according to the embodiments of the present application;

[0061] Figure 3According to the multiple reaction mode chromatogram in the examples of this application;

[0062] Figure 4 According to the main fragment ion attribution diagram of the secondary mass spectrum in the embodiment of the present application;

[0063] Figure 5 According to the specificity investigation of the polypeptides (PP1, PP6, PP12) in the examples of this application;

[0064] Figure 6 According to the test results of the batch of blind-ringed earthworms in the examples of this application. DETAILED DESCRIPTION

[0065] To make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the present application is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples that do not specify specific conditions are usually based on normal conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0066] 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 the 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 the present application.

[0067] Example 1

[0068] In this embodiment, a screening method for developing characteristic peptides of earthworms is developed. The method comprises the following steps:

[0069] 1. Obtain peak intensity data

[0070] Based on the high-resolution data of four genuine earthworms and four counterfeit earthworms (MM, Amynthas amis, AA, A. carnosus, AC, and P. tschiliensis, PT) and quality control samples (all samples were mixed in equal proportions), the ion information was extracted using Progenisis QI software (Waters, Redmond, WA, USA). Select multivalent ions [M+2H] 2+ 、[M+3H] 3+ 、[M+4H] 4+ 、[M+5H] 5+ , a data table of retention time, mass-to-charge ratio, and normalized peak intensity was obtained, which contained a total of 19433 ions, mainly 2-4 valent ions.

[0071] 2. Principal Component Analysis

[0072] The quality control data were analyzed using the "80% rule" and "30% variation" and the data that did not meet the rules were eliminated, leaving 14,895 ions. The processed data list was imported into SIMCA-P 14.1 software (Umetrics AB, Umea, Sweden) for PCA (unsupervised pattern recognition method) analysis. The results are shown in Figure 1 , Shanghai dragon and its common counterfeits are grouped together, including the Pheretima PP (2 batches).

[0073] 3. Preliminary screening of differential ions

[0074] Two batches of PP were used as a group, and Shanghai Dragon (PV and PG) and common counterfeit products were used as a group. A t-test was conducted, and the data with a p-value greater than 0.05 were deleted. Then, the average value of the two groups of data was used as the quotient, and the ions with high content in PP (ratio ≥ 100) were selected and sorted according to peak intensity. 7 The ions were selected as candidate characteristic peptide ions, and 42 differential ions were finally preliminarily screened out.

[0075] 4. Verification of differential ion specificity

[0076] The daughter ions of differential ions were screened through secondary mass spectrometry analysis, and a multiple reaction monitoring method was constructed through retention time prediction. Liquid chromatography-tandem triple quadrupole mass spectrometry was used to verify the specificity of differential ions.

[0077] 4.1 Selecting product ions

[0078] By analyzing the secondary high-resolution mass spectra, the daughter ions with good response were selected. The representative spectra are shown in Figure 2 , two product ions are selected for each differential ion.

[0079] 4.2 Retention time prediction

[0080] Preparation method of test sample: Take 0.3g of the powder of this product, add protein lysis solution [weigh 6.06g of tris(hydroxymethyl)aminomethane, add appropriate amount of water to stir and dissolve, and dilute to 1000ml, adjust the pH value to 8.8 with 2mol / L hydrochloric acid solution, then add 40g of sodium dodecyl sulfate, stir to dissolve. Before use, weigh an appropriate amount of dithiothreitol, add the above solution to dissolve, prepare a dithiothreitol solution with a concentration of 20mmol / L, that is, 15ml, place it at 80℃ for ultrasonic treatment (power 500W, frequency 40kHz) for 1 hour, let it stand at room temperature, centrifuge for 20 minutes (12000 revolutions per minute), take 200μl of the supernatant, add 20μl of 0.5mol / L iodoacetamide solution, place it at room temperature in a dark place for 45 minutes, add 900μl of acetone (precooled at-20℃), mix well, precipitate at-20℃ for 4 hours, centrifuge at 4℃ for 20 minutes (12000 revolutions per minute), discard the supernatant, wash the residue with acetone (precooled at-20℃) twice, 200μl each time, evaporate the acetone, add 100μl of 8mol / L urea solution to the residue, dissolve it by ultrasonication (temperature not exceeding 37℃), add 800μl of 1% ammonium bicarbonate solution, and mix well. Add 20 μl of trypsin solution (1 mg / ml), shake well, and perform enzymolysis at 37°C for 18 hours.

[0081] Data acquisition method: Agilent 1290 ultra-high performance liquid chromatograph-Agilent 6495 triple quadrupole mass spectrometer was used to collect data, the chromatographic column was CORTECS T3 C18 (2.1 mm × 10 cm, 2.7 μm), the mobile phase was acetonitrile (A)-0.1% formic acid aqueous solution (B), gradient elution, 0-15 minutes, 13% → 32% A, injection volume 5 μl, flow rate 0.4 ml / min. 2 Scan mode, scanning range m / z 350-2000, positive ion mode, analysis of Pheretima sphenanthera samples.

[0082] Retention time prediction: From the 42 ions, representative ions were selected according to the retention time distribution, and the actual retention time of triple quadrupole mass spectrometry was recorded through the extracted ion chromatogram (EIC), and the linear relationship was investigated with the actual retention time of the high-resolution mass spectrometry of the corresponding ions, Y = 3.9906X + 23.3 (R 2 =0.9682)(Y is the actual retention time of high-resolution mass spectrometry, X is the actual retention time of triple quadrupole mass spectrometry), the triple quadrupole mass spectrometry theoretical retention time was calculated by the equation, and the results (Table 1) showed that the maximum deviation was 1.06min compared with the actual detection time. The theoretical retention time of the remaining 32 ions was calculated using the above equation, and a dynamic multiple reaction monitoring (DMRM) method was constructed, with a time deviation of 4min (i.e., the deviation before and after the theoretical retention time was 2min).

[0083] Table 1 Calculated predicted retention times of representative ions

[0084]

[0085] 4.3 Differential ion verification

[0086] The constructed DMRM method is shown in Table 2. The DMRM methods for analyzing Pheretima annuli (PA), Pheretima vulgaris (PV), Pheretima viridis (PG), Pheretima fasciatum (PP), Pheretima baoningensis (MM), Pheretima zhiliensis (PT), Pheretima succulenta (AC), Pheretima amerikani (AA), and Pheretima californica (MC) are shown in the overlay of the extracted ion chromatograms. Figure 3 The results showed that 18 ions were only detected in Pheretima ciliata (PP1, PP2, PP6, PP7, PP8, PP12, PP14, PP21, PP23, PP24, PP25, PP26, PP28, PP32, PP34, PP35, PP37, and PP38), with good specificity.

[0087] Table 2DMRM method

[0088]

[0089]

[0090]

[0091] 5. Peptide sequence identification

[0092] 5.1 Peptide sequence analysis

[0093] Using PEAKS Xpro 10.0 (Bioinformatics Solutions Inc.) software used the Denovo de novo sequencing method to identify the amino acid sequence of the peptide. The nonspecific mode was selected, the precursor ion mass deviation was set to 10 ppm, and the fragment ion mass deviation was set to 0.02 Da. PTMs setting parameters: variable modifications selected oxidation modification (oxidation, +15.99 Da) and N-terminal acetylation (acetylation, +42.01 Da). Other parameters were set by default. For the identified peptides, the false discovery rate (FDR) was <1%, the de novo score was >90, and for proteins, the FDR was <1%, with at least 1 unique peptide.

[0094] From the 18 ions, three ions with high response intensity and no modification were selected, and the amino acid sequences of the corresponding ions were checked from the identification results as shown in Table 3. The main fragment ions of the secondary mass spectrum are shown in Figure 4 There are three possible polypeptide sequences of PP6.

[0095] Table 3 Peptide sequence identification results

[0096] name Retention time (min) m / z Amino acid sequence serial number Sequence length PP1 46.5 700.3279 SIWTDTESSAFR SEQ ID NO.1 12 PP6-1 36.7 701.3321 FASQSGMASFNVR SEQ ID NO.2 13 PP6-2 36.7 701.3321 FASAGAGMTSFQAR SEQ ID NO.3 14 PP6-3 36.7 701.3321 FASQSGMTFGNVR SEQ ID NO.4 13 PP12 49.1 657.8671 SIVIAGEVEELR SEQ ID NO.5 12

[0097] 5.2 Synthesis of reference substances and verification of peptide accuracy

[0098] The product was commissioned to be synthesized by Gill Biochemical (Shanghai) Co., Ltd., and the purity is greater than 95%.

[0099] Preparation of reference substance stock solution: Take appropriate amount of the above 5 polypeptide reference substances, dissolve them in 1% ammonium bicarbonate solution to prepare solutions with a concentration of 10μg / ml, and obtain each reference substance stock solution. Preparation of solvent reference substance solution: Take appropriate amount of the stock solutions (PP1, PP6-1, PP12) in the same volumetric flask, dilute them with 1% ammonium bicarbonate solution to prepare a mixed reference substance solution with a concentration of 100ng / ml; take another stock solution (PP6-2, PP6-3), dilute them with 1% ammonium bicarbonate solution to prepare a single standard solution with a concentration of 100ng / ml. Preparation of matrix reference solution: Take appropriate amount of stock solution (PP1, PP6-1, PP12) in the same volumetric flask, dilute it with the test solution of Pheretima sphenanthera to prepare a mixed solution with a concentration of 100 ng / ml; take another stock solution (PP6-2, PP6-3), dilute it with the test solution of Pheretima sphenanthera to prepare a single-label matrix solution with a concentration of 100 ng / ml.

[0100] Take 5μl of the above-mentioned reference solution, reference matrix solution and Pheretima sphenanthera test solution respectively, and analyze according to "4.2 Data Collection Method". The results show that the reference solution presents corresponding daughter ion chromatographic peaks. The retention time of the solvent reference solution of polypeptides PP1, PP6-3, and PP12 is slightly different from that of the test solution (difference of 0.1-0.5 minutes). The corresponding daughter ions of the matrix reference solution present a single chromatographic peak, and the retention time is basically consistent with that of the test solution. The corresponding daughter ions of the matrix reference solution of polypeptides PP6-2 and PP6-1 present two chromatographic peaks respectively, which can be seen that they are not the polypeptide sequences in the test solution.

[0101] 6. Identification method of peptides from Pheretima sutchuenensis

[0102] Based on the specificity, response intensity, stability and other factors of the polypeptide, PP6-3 was finally selected as the quality control indicator to establish an identification method for Pheretima pubescens.

[0103] 6.1 Chromatographic and mass spectrometric conditions

[0104] Data were collected using an Agilent 1290 ultra-high performance liquid chromatograph-Agilent 6495 triple quadrupole mass spectrometer, using a CORTECS T3 C18 column (2.7 μm, 2.1×100 mm); acetonitrile was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, the elution gradient was 0-15 min, 13%-32% A; the injection volume was 5 μL; the flow rate was 0.4 mL / min. Electrospray positive ionization mode (ESI + ), dryer temperature 200 °C, gas flow rate 13.0 L / min, nebulizer 35 psi, sheath gas flow rate 12 L / min, fragmentor voltage 380 V, multiple reaction monitoring (MRM) was performed, and the mass-to-charge ratios of m / z 701.3 (doubly charged)→968.5 (quantitative ion) and m / z 701.3 (doubly charged)→881.4 (qualitative ion) were selected as the detection ion pairs.

[0105] 6.1 Specificity inspection

[0106] For details of the experiment, see "4.3 Differential Ion Verification". Figure 5 , the three polypeptide ions were only detected in Pheretima ciliata, but not in other original species, indicating that the method has good specificity.

[0107] 6.2 Repeatability study

[0108] Take the earthworm sample, prepare 6 samples in parallel according to the test solution preparation method, inject and measure separately, record the retention time and peak intensity of the quantitative reaction ion pair of polypeptide PP6-3, calculate RSD, and the results are shown in Table 4. The retention time RSD is less than 1.0%, and the peak intensity RSD is less than 5.0%, indicating that the method has good repeatability.

[0109] 6.3 Stability investigation

[0110] The test solution of Pheretima ciliata was taken and sampled at 0h, 4h, 8h, 12h, 16h and 24h, respectively, and the retention time and peak intensity of the quantitative reaction ion pair of polypeptide PP6-3 were recorded, and the RSD was calculated. The results are shown in Table 4. The retention time RSD was less than 2.0%, and the peak intensity RSD was less than 5.0%, indicating that the test solution was basically stable within 24h at room temperature.

[0111] Table 4 Repeatability and stability test results

[0112]

[0113] 6.4 Sample determination

[0114] A total of 6 batches of samples were analyzed using the above method, and the results all showed chromatographic peaks at m / z 701.3→968.5 and m / z 701.3→881.4, see Figure 6 , indicating that the sources of the 6 batches of samples were all Pheretima ciliata.

[0115] The variety identification was carried out according to the guidelines for the molecular identification of Chinese herbal medicines using DNA barcodes in 9107 of the 2020 edition of the Chinese Pharmacopoeia. PCR amplification was performed using COI universal primers, and the amplified products were entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. for bidirectional sequencing. The sequencing results were spliced ​​and compared with the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and the Chinese herbal medicine DNA barcode identification database (http: / / www.gpgenome.com / blast). Results The six batches of samples were all from Pheretima pectinifera (i.e. Amynthas pectinifera), which proved the accuracy of the characteristic peptide method.

[0116] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present application, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present application.

Claims

1. A method for identifying Pheretima sphenanthera, characterized in that: The method comprises the steps of: (1) Providing a sample of earthworm to be tested; (2) Detecting the content of the polypeptide shown in SEQ ID NO.4 (PP6-3) in the earthworm sample, and determining whether the earthworm sample contains the polypeptide shown in SEQ ID NO.4 (PP6-3) by analyzing whether the earthworm sample contains the polypeptide shown in SEQ ID NO.4 (PP6-3).

2. The method according to claim 1, characterized in that The earthworm sample is a medicinal earthworm.

3. The method according to claim 1, characterized in that In the step (2), a liquid chromatography-tandem triple quadrupole mass spectrometer is used to detect the sample to be tested, and the mass spectrometry conditions of the liquid chromatography-tandem triple quadrupole mass spectrometer include: setting m / z 701.3 (doubly charged) → 968.5 as a quantitative ion, and setting m / z 701.3 (doubly charged) → 881.4 as a qualitative ion for detection.

4. The method according to claim 3, characterized in that The mass spectrometry conditions of the liquid chromatography tandem triple quadrupole mass spectrometer also include: Electrospray positive ionization mode (ESI + ), Detection mode: Multiple reaction monitoring (MRM).

5. The method according to claim 4, characterized in that The mass spectrometry conditions of the liquid chromatography tandem triple quadrupole mass spectrometer also include: Dryer temperature 180-220°C (e.g. 200°C); Gas flow rate 10-15 L / min (e.g. 13.0 L / min); Atomizer 30-40 psi (e.g. 35 psi); Sheath gas flow rate 10-15 L / min (e.g. 12 L / min); The fragmentation voltage is 350-400V (eg 380V).

6. The method according to claim 3, characterized in that The chromatographic conditions of the liquid chromatography-tandem triple quadrupole mass spectrometer include: using acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B for gradient elution; Preferably, the chromatographic conditions of the liquid chromatography-tandem triple quadrupole mass spectrometer include: the gradient elution procedure is: within 0 to 15 minutes, the volume proportion of mobile phase A is uniformly increased from 13% to 32%, and the volume proportion of mobile phase B is uniformly reduced from 87% to 68%.

7. The method according to claim 6, characterized in that The chromatographic conditions of the liquid chromatography tandem triple quadrupole mass spectrometer also include: Use a C18 column (e.g., CORTECS T3 C18 column (2.7 μm, 2.1 × 100 mm); Injection volume: 5 μL; Flow rate: 0.4 mL / min.

8. A characteristic polypeptide for identifying Pheretima sphenanthera, characterized in that: The amino acid sequence of the characteristic polypeptide is selected from at least one of SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.

5.

9. Use of the characteristic polypeptide as claimed in claim 8 in identifying earthworm samples.

10. A method for screening characteristic polypeptides of earthworms, characterized in that: The method comprises the steps of: S1, obtain the original data table of earthworm samples; The method comprises the following steps: using high-resolution mass spectrometry to analyze authentic earthworms, counterfeit earthworms and quality control samples, extracting ion information to obtain an original data table of earthworm samples containing retention time, mass-to-charge ratio and normalized peak intensity information; S2, remove the non-compliant data in the original data table, and use PCA analysis to analyze the original data to screen the target earthworm sample peaks. S3, obtaining the differential ions of the target earthworm sample by comparing the difference between the target earthworm sample peak and other peaks; S4, screening differentiated ions with strong specificity by performing specificity analysis on the differentiated ions; S5, by De novo sequencing, the peptide sequences of the highly specific differential ions described in S4 are resolved; S6, verify the accuracy of the polypeptide sequence.

11. The method according to claim 10, characterized in that The characteristic polypeptide of earthworm is the characteristic polypeptide of Pheretima sphenanthera. The method comprises the steps of: S1, obtain the original data table of earthworm samples from various sources; The method comprises the following steps: using a high-resolution mass spectrometer to analyze various genuine earthworms, counterfeit earthworms and quality control samples, extracting ion information to obtain an original data table of earthworm samples containing retention time, mass-to-charge ratio and normalized peak intensity information; S2, remove the non-compliant data in the original data table, and use PCA analysis to analyze the original data to screen the peaks of Pheretima sphenanthera samples; S3, using Pheretima sphenanthera as the first group and L. humilis and its other fakes as the second group for t-test analysis, several differential screening ions were obtained; S4, screening the Pheretima sphenanthera differentiated ions with strong specificity by performing specificity analysis on the plurality of Pheretima sphenanthera differentiated ions; S5, by De novo sequencing, the peptide sequence of the highly specific differential ions of Pheretima sphenanthera described in S4 was analyzed; S6, verifying the accuracy of the polypeptide sequence, that is, obtaining a polypeptide characteristic of Pheretima pubescens.

Images