Differential characteristic polypeptide library for identifying different medicinal parts of bufo bufo gargarizans and application of differential characteristic polypeptide library

Abstract

The invention belongs to the technical field of biology, and particularly relates to a differential characteristic polypeptide library for identifying different medicinal parts of bufo bufo gargarizans and application of the differential characteristic polypeptide library. In the prior art, toad venom is different from toad skin, toad meat and toad bone in protein components, but detection of protein macromolecules is tedious, and conversion from a method to a standard is not facilitated. The polypeptide is used as an index component to identify and analyze the venenum bufonis and dried toad medicinal materials, the specificity is high, the stability is good, the interference of the processing process is avoided, multiple medicinal materials can be identified at the same time, and the advantages are obvious.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a differential characteristic polypeptide library for identifying different medicinal parts of Bufo bufo gargarizans and an application thereof. Background Art

[0002] Chinese giant toad ( bufo gargarizans ) is a commonly used medicinal animal in clinical Chinese medicine. Toads are "spicy, cool, and poisonous. They are in the heart, liver, spleen, and lung meridians. They can "detoxify, disperse stagnation, eliminate stagnation and promote diuresis, kill insects and eliminate malnutrition. They are mainly used to treat carbuncle, furuncle, back rash, scrofula, malignant sores, fatigue, swelling, edema, infantile malnutrition, tetanus, and chronic cough and asthma". Ancient and modern prescriptions have widely used them to remove toxins and reduce swelling, relieve pain and kill insects, strengthen the heart and promote diuresis. In modern clinical practice, they are used to fight tumors, relieve pain, promote diuresis and reduce swelling, and enhance the body's immunity. The whole body of the toad can be used as medicine, and commonly used ones are toad venom and toad skin.

[0003] In recent years, due to the continuous expansion of clinical applications of toad venom and its high price, commercial toad venom is mixed, adulterated and counterfeited. Traditional identification methods such as property identification, microscopic identification and physical and chemical identification cannot effectively distinguish toad venom from toad skin, toad meat and toad bones, and have limitations. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a differential characteristic polypeptide library for identifying different medicinal parts of Bufo bufo gargarizans.

[0005] The present invention also provides the use of the differential characteristic polypeptide library in distinguishing and identifying toad venom and toad skin, toad meat and toad bone.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The invention provides a differential characteristic polypeptide library for identifying different medicinal parts of Bufo bufo gargarizans. The characteristic polypeptide library consists of sequences such as SEQ ID NO.1-29.

[0007] The present invention also provides the use of the above differential characteristic polypeptide library in rapidly distinguishing and identifying different medicinal parts of Bufo bufo gargarizans, comprising the following steps: (1) Sample preparation: accurately weigh the sample powder, add protein lysis buffer, perform ultrasonic extraction, centrifuge and collect the supernatant to obtain the protein sample solution; (2) Reductive alkylation: Take a protein sample solution, add dithiothreitol (DTT) solution, and place it at room temperature for a period of time; then add iodoacetamide (IAA) solution, and place it in the dark at room temperature for a period of time; then add ammonium bicarbonate solution to obtain a reduced alkylated protein solution; (3) Enzyme cleavage and desalting: Add trypsin to the reduced alkylated protein solution and digest overnight; then add FA to terminate the enzymatic cleavage reaction and perform desalting.

[0008] (4) Triple quadrupole liquid chromatography-mass spectrometry was used for identification.

[0009] Furthermore, in step (1), the ratio of the sample powder to the protein lysate is 25 mg:1 mL; the composition of the protein lysate is: 21.0 g urea, 7.60 g thiourea, 50 μL Triton X-100, and 50 mM ammonium bicarbonate solution is added to make the volume to 50 mL.

[0010] Furthermore, in step (1), the ultrasonic extraction time is 30 minutes; the centrifugation is at 4°C and 13000 r∙min. -1 Centrifuge for 15 minutes.

[0011] Furthermore, in step (2), the concentration of dithiothreitol (DTT) in the protein sample solution is 10 mM; the concentration of dithiothreitol (DTT) solution is 1 M; the concentration of iodoacetamide (IAA) in the protein sample solution is 50 mM; the concentration of iodoacetamide (IAA) solution is 1 M; the amount of ammonium bicarbonate solution added is to reduce the urea concentration in the protein sample solution to below 1 M; the concentration of ammonium bicarbonate solution is 50 mM.

[0012] Furthermore, in step (2), the time of being placed at room temperature is 3 hours; and the time of being placed in a dark place at room temperature is 1 hour.

[0013] Furthermore, in step (3), in the reduced alkylated protein solution, the mass ratio of protein to trypsin is 100:1; and the mass concentration of FA in the reduced alkylated protein solution is 0.5%.

[0014] Furthermore, in step (3), the enzyme digestion is carried out at 37°C overnight; the desalting is carried out using Sep-PakC 18 column.

[0015] Furthermore, in step (4), the liquid chromatography conditions are: using Thermo Acclaim PepMap C 18 The desalting and enrichment were performed on a Thermo Acclaim PepMap C column (100 μm × 3.5 cm, 5 μm). 18 Separation on a column (75 μm×15 cm, 3 μm), mobile phase: 0.1% (v / v) formic acid in water (A) and 0.1% (v / v) formic acid in acetonitrile (B), gradient elution, flow rate: 300 nL·min -1, injection volume: 1 µL; the gradient elution program was: 0~1 min, 99%→94%A; 1~96 min, 94%→78%A; 96~113 min, 78%→70%A; 113~117 min, 70%→5%A; 117~120 min, 5%A.

[0016] Furthermore, the mass spectrometry conditions are as follows: analysis in positive ion mode, spray voltage: 2.1 kV, ion transmission capillary temperature: 275 °C, S-Lens transmission efficiency: 60%; primary mass spectrometry uses Orbitrap as a mass analyzer, with a resolution of 60000 and an acquisition range of 350-1550 ( m / z ); The secondary mass spectrometer also uses Orbitrap as the mass analyzer, the scanning mode is Data-Dependent MSn Scan mode, and the fragmentation mode is HCD mode.

[0017] In the prior art, there are differences in protein composition between toad venom and toad skin, toad meat, and toad bone, but the detection of protein macromolecules is relatively cumbersome, which is not conducive to the conversion of methods into standards. The present invention uses polypeptides as index components to identify and analyze toad venom and dried toad medicinal materials, which has strong specificity, good stability, is not affected by the processing process, and can identify multiple medicinal materials at the same time, which has obvious advantages.

[0018] The beneficial effects of the present invention are: (1) The identification method provided by the present invention can simultaneously identify and authenticate toad venom, toad skin, toad meat, and toad bones, with good specificity and high efficiency; (2) The characteristic peptide library provided by the present invention has strong specificity. In the subsequent application process, the detection method is simple and fast. The quantitative determination method of toad venom and the inspection method of dried toad components in toad venom are established, which provides method support for the quality control of toad venom and improves the accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 BSA standard curve; Figure 2 Principal component analysis of toad-derived medicinal materials; Figure 3 OPLS-DA graph (A) and score graph (B) comparing toad venom and toad skin; Figure 4 OPLS-DA graph (A) and score graph (B) comparing toad venom and toad meat; Figure 5 OPLS-DA graph (A) and score graph (B) comparing toad venom and toad bone; Figure 6NNAYDINEER validation process of peptide sequences (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Figure 7 FLEYGHR validation process of peptide sequences (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Figure 8 Peptide sequence NVWSPDEK verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig. 9 Peptide sequence KGQDFEVR verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.10 The peptide sequence FNEGHVLF verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.11 Peptide sequence SAWPLLPGK verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.12 Peptide sequence ASEPAGPADEVK verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.13 Peptide sequence SAWPLF(sub L)PGK verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results, D: Nano LC-MS / MS verification results); Fig.14Verification process of the peptide sequence GVAGPPGAVGASGK (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.15 The validation process of the peptide sequence GTVGLPGQR (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.16 Peptide sequence ISNIPDEYFQGFK verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.17 Peptide sequence GEIGPAGS(sub N)TGPTGAAGGR verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results, D: Nano LC-MS / MS verification results); Fig.18 Verification process of the peptide sequence SGEPGAAGPP(+15.99)GPP(+15.99)GEK (A: prediction results of Peaks Studio software, B: homology analysis results, C: verification results of AB SCIEX 6500+ liquid chromatography-mass spectrometry, D: verification results of Nano LC-MS / MS); Fig.19 Verification process of peptide sequence GFPGLP(+15.99)GPNGEPGK (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results, D: Nano LC-MS / MS verification results); Fig. 20 Verification process of the peptide sequence GESGPAGPP(+15.99)GAPGAPGAP(+15.99)GPAGPAGK (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results, D: Nano LC-MS / MS verification results); Fig.21Peptide sequence QLFEGLSR verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig. 22 Peptide sequence LLAGQSIISSAR validation process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.23 Peptide sequence YEVTTLR verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.24 Peptide sequence DLEEATLQHEATAS(sub A)ALR verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results, D: Nano LC-MS / MS verification results); Fig.25 The validation process of peptide sequence VTFQLPA(sub S)ER (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results, D: Nano LC-MS / MS verification results); Fig.26 Peptide sequence SEEELAEC (+57.02) FR verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results, D: Nano LC-MS / MS verification results); Fig. 27 VDMTTLR validation process of peptide sequence (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.28 The validation process of the peptide sequence VGLLNYASSVK (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋Liquid chromatography-mass spectrometry verification results) Fig.29 Peptide sequence YPIVNPR verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.30 Peptide sequence AGWLSDGSVR verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.31 Peptide sequence YPIVTPR verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.32 Peptide sequence GLAGPQGPR verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results) Fig.33 Peptide sequence TPQAFVC (+57.02) LPR verification process (A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results, D: Nano LC-MS / MS verification results); Fig.34 Peptide sequence ETWVDAENLC(+57.02)R verification process A: Peaks Studio software prediction results, B: homology analysis results, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification results, D: Nano LC-MS / MS verification results); Fig.35 This is a typical diagram of the identification results of toad venom (taking the 461.2→661.3 ion pair as an example); Fig.36 Typical graph of toad skin screening results (taking the 718.8→867.4 ion pair as an example); Fig.37 Typical graph of toad meat screening results (taking the 619.3→818.4 ion pair as an example); Fig.38 Typical graph of toad bone determination results (taking the 418.2→612.4 ion pair as an example). DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0021] The sample information used in the present invention is shown in Table 1.

[0022] Table 1 Sample information Analysis software: SIEVE 2.2, SIMCA 14.1 (32-bit).

[0023] Example 1 1.1 Experimental methods 1.1.1 Sample preparation Take about 250 mg of sample powder, weigh accurately, add 10 mL of protein lysis buffer (urea 21.0 g, thiourea 7.60 g, Triton X-100 50 μL, add 50 mM ammonium bicarbonate solution to 50 mL), extract by ultrasonic for 30 min, leave overnight, centrifuge at 4 °C for 15 min (13000 r∙min -1 ), take the supernatant and obtain the protein sample solution for subsequent experiments.

[0024] 1.1.2 Protein concentration determination The protein concentration was determined using a modified Bradford protein concentration assay kit.

[0025] Preparation of standard solution: Pipette 1.00 mg·mL -1 0, 50, 100, 150, 200, 250, 300 μL of BSA standard protein were placed in a 2 mL centrifuge tube, and then 1000, 950, 900, 850, 800, 750, 700 μL of pure water were added to dilute them, and the concentrations were 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30 mg·mL -1 BSA standard protein solution.

[0026] Accurately pipette 20 μL of BSA standard protein and toad venom protein solution of different concentrations into different wells of a 96-well plate, then add 200 μL of Bradford working solution into the wells, and measure the absorbance at 595 nm with an enzyme-labeled instrument.

[0027] 1.1.3 Reductive alkylation According to the results of the protein solution concentration determination, take 250 μg of protein sample, add an appropriate amount of 1 M dithiothreitol (DTT) solution to make the final concentration of DTT 10 mM, and place it at room temperature for 3 hours. Then add an appropriate amount of 1 M iodoacetamide (IAA) solution to make the final concentration of IAA 50 mM, and place it in the dark at room temperature for 1 hour. Then add 50 mM ammonium bicarbonate solution to reduce the urea concentration to below 1 M.

[0028] 1.1.4 Protease cleavage and desalting Trypsin was added at a ratio of 100:1 and digested in a constant temperature and humidity chamber at 37°C overnight (12 h). The next day, appropriate FA was added to make the final FA concentration 0.5% (mass concentration) to terminate the digestion reaction. 18 Desalting column.

[0029] 1.1.5 Nano analysis conditions Chromatographic conditions: Thermo Acclaim PepMap C 18 The desalting and enrichment were performed on a Thermo Acclaim PepMap C column (100 μm × 3.5 cm, 5 μm). 18 The samples were separated on a column (75 μm×15 cm, 3 μm). The mobile phases were 0.1% (v / v) formic acid in water (A) and 0.1% (v / v) formic acid in acetonitrile (B). The elution was performed by gradient elution (0–1 min, 99%→94%A; 1–96 min, 94%→78%A; 96–113 min, 78%→70%A; 113–117 min, 70%→5%A; 117–120 min, 5%A). The flow rate was 300 nL min -1 , injection volume: 1 µL.

[0030] Mass spectrometry conditions: analysis in positive ion mode, spray voltage: 2.1 kV, ion transfer capillary temperature: 275 °C, S-Lens transmission efficiency: 60%; primary mass spectrometry using Orbitrap as mass analyzer, resolution 60000, acquisition range 350-1550 ( m / z ); The secondary mass spectrometer also uses Orbitrap as the mass analyzer, the scanning mode is Data-Dependent MSn Scan mode, and the fragmentation mode is HCD mode.

[0031] 1.1.6 Peaks Search Peaks Studio software (version 8.5) was used to identify peak components in combination with the Bufo bufo gargarizans database downloaded from NCBI. The parameters were set as follows: trypsin was selected with a maximum of 6 missed cuts. Oxidation (+15.99), hydroxylation (+15.99), deamidation (+0.98), and acetylation of the N-terminus of the protein (+42.01) were specified as variable modifications. The maximum precursor ion tolerance was 15 ppm, the fragment ion tolerance was 0.02 Da, the filter charge was set to 2 to 8, and all other parameters were set by default. The FDR (false discovery rate) for peptides and proteins was set to 1%.

[0032] 1.1.7 Chemometric analysis Based on the peptide spectrum, SIEVE and SIMCA software were used to screen mass spectrometry characteristic ions to find mass spectrometry information to distinguish toad venom from toad skin, toad meat and toad bone.

[0033] 1.1.7.1 Mass spectrometry data preprocessing The data collected by high-resolution mass spectrometry were imported into SIEVE 2.2 software, and the toad venom sample was selected as the reference sample. All samples were peak aligned and framed, and the precise mass-to-charge ratio, retention time, peak area and other information of the ions were extracted. Each ion pair was numbered for subsequent chemometric analysis.

[0034] 1.1.8 Specificity Verification Method The candidate potential characteristic ions must be verified by mass spectrometry to determine their actual presence in different samples of toad venom, toad skin, toad meat, and toad bone to determine whether they are exclusive. ＋ The multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometry was verified by chromatographic behavior, and the specificity of the characteristic ions was confirmed based on the presence or absence of chromatographic peaks and responses of potential characteristic ions in different toad venom, toad skin, toad meat and toad bone samples.

[0035] Chromatographic method: Column Agilent Eclipse C 18 The samples were separated by chromatography (2.1×100 mm, 1.8 µm) with an injection volume of 5 µL and a flow rate of 0.3 mL min -1 , the column was kept at 43 °C. The mobile phase consisted of A (0.1% formic acid in water) and B (acetonitrile solution). Elution gradient: 0-20 min, 3%→25%B, 20-21 min, 25%→90%B, 21-24 min, 90%B, 24.1-30 min, 3%B.

[0036] Mass spectrometry method: The LC-MS / MS system was controlled using Analyst Software. The parameters were set as follows: the mode was set to electrospray ionization (ESI) and positive ion MRM mode, and the sheath gas flow rate was 46 L∙h -1 , Auxiliary gas flow rate: 850 L∙h -1 , Spray Voltage: 3.5 kV, Source temperature: 150 °C, Auxiliary gas temperature: 400 °C. Cone Voltage: 30 V, Collision voltage: 35 V.

[0037] Example 2 Result Analysis 1.1 Chemometric results Figure 2 It can be seen that there is an obvious separation trend among toad venom, toad skin, toad meat and toad bone, indicating that there are differences in protein and polypeptide components of toad-derived medicinal materials.

[0038] The data analysis results are shown in Figure 3 , Figure 4 , Figure 5 , each point represents an accurate mass-retention time data pair. In the two-dimensional data analysis diagram, when comparing toad venom with toad skin, toad meat, and toad bone, they all show good separation effects. In the S curve of the corresponding score diagram, the points at both ends of the curve are more likely to be potential peptide markers.

[0039] Through chemometric analysis, 83 potential characteristic ions were screened out to distinguish toad venom from toad skin, toad meat and toad bones, and 97, 60 and 41 potential characteristic ions were screened out to distinguish toad skin, toad meat and toad bones from toad venom respectively. After final confirmation, 29 characteristic peptides were obtained.

[0040] 2.1 Experimental Materials During the detection process, some biomarkers were synthesized, and detailed information is shown in Table 10.

[0041] Table 10 Information table of peptide biomarker reference substances 2.2 Experimental methods 2.2.1 Sample preparation Preparation of synthetic peptides: Accurately weigh 1 mg of synthetic peptide, add 1 mL of water, and shake until completely dissolved to obtain a reference substance concentration of 1 mg mL -1 of stock solution.

[0042] Preparation of spiked samples: According to the response values ​​of synthetic peptides and samples, the two were mixed in proportion to prepare spiked samples.

[0043] The reference stock solution, test solution and spiked solution were diluted to appropriate concentrations and injected for analysis.

[0044] 2.2.2 Analysis conditions AB SCIEX 6500 ＋ The chromatographic and mass spectrometric conditions of the liquid chromatography-mass spectrometer are the same as those in Example 1.

[0045] The chromatographic and mass spectrometric conditions of EASY-nLC 1000 liquid chromatography-Orbitrap Fusion combined high-resolution mass spectrometer were the same as those in Example 1.

[0046] 2.3 Experimental Results There are 7 differential peptide sequences without modification in Toad Venom, and 1 differential peptide sequence with modification, as shown in the following figure: Figure 6 Shown There are 3 differential peptide sequences in toad skin without modification, and 4 differential peptide sequences with modification. The confirmed sequences are as follows Figure 7-10 shown.

[0047] There are 3 differential peptide sequences in toad meat without modification, and 3 differential peptide sequences with modification. The confirmed sequences are as follows Figure 11-13 shown.

[0048] There are 6 differential peptide sequences in toad bones without modification, and 2 differential peptide sequences with modification. Fig.14 and Fig.15 shown.

[0049] Example 3 1. Sample preparation According to the prescribed amount of toad venom in the preparations (Xiongdan Jiuxin Pills, Liuling Pills, Houzheng Pills, Niuhuang Xiaoyan Tablets), weigh an equivalent amount of the preparations and prepare a sample solution according to the sample preparation method in 1.1 Experimental Method in Example 1.

[0050] 2 Analysis conditions Chromatographic method: Column: Agilent HSS C 18 (2.1×100 mm, 1.8 µm), column temperature 35 °C, flow rate 0.3 mL∙min -1 , mobile phase 0.1% formic acid in water (A) and acetonitrile (B), gradient elution 0-20 min, 3-25% B, 20-21 min, 25-90% B. Injection volume 5 µL.

[0051] Mass spectrometry method: electrospray ionization source (ESI), MRM monitoring in positive ion mode: Scan range m / z: 100~1250; IS: 5500 V, TEM: 550℃; CUR: 30 psi; GS1: 50psi; GS2: 50 psi; CAD: 8.

[0052] 3 Experimental Results Toad venom peptide signals were detected in all four preparations, and toad skin components were also detected in all of them. Toad meat peptide signals were detected in Xiongdan Jiuxin Pills and Niuhuang Xiaoyan Tablets. The results are as follows: Figure 35-38 shown.

Claims

1. A differential characteristic polypeptide library for identifying different medicinal parts of Bufo bufo gargarizans, characterized in that: The characteristic polypeptide library consists of sequences such as SEQ ID NO.1-29; specifically: 。 2. An application of the differential characteristic polypeptide library as claimed in claim 1 in rapid differentiation and identification of different medicinal parts of Bufo bufo gargarizans, characterized in that: The following steps are involved: (1) Sample preparation: accurately weigh the sample powder, add protein lysis buffer, perform ultrasonic extraction, centrifuge and collect the supernatant to obtain the protein sample solution; (2) Reductive alkylation: Take a protein sample solution, add dithiothreitol (DTT) solution, and place it at room temperature for a period of time; then add iodoacetamide (IAA) solution, and place it in the dark at room temperature for a period of time; then add ammonium bicarbonate solution to obtain a reduced alkylated protein solution; (3) Enzyme cleavage and desalting: Add trypsin to the reduced alkylated protein solution and allow for overnight enzymatic cleavage. Then add formic acid (FA) to terminate the enzymatic cleavage reaction and perform desalting. (4) Triple quadrupole liquid chromatography-mass spectrometry was used for identification.

3. The use according to claim 2, characterized in that: In step (1), the ratio of the sample powder to the protein lysate is 25 mg:1 mL; the composition of the protein lysate is: 21.0 g urea, 7.60 g thiourea, 50 μL Triton X-100, and 50 mM ammonium bicarbonate solution is added to make the volume to 50 mL.

4. The use according to claim 2 or 3, characterized in that: In step (1), the ultrasonic extraction time is 30 minutes; the centrifugation is at 4°C and 13000 r∙min. -1 Centrifuge for 15 minutes.

5. The use according to claim 2, characterized in that: In step (2), the concentration of dithiothreitol (DTT) in the protein sample solution is 10 mM; the concentration of dithiothreitol (DTT) solution is 1 M; the concentration of iodoacetamide (IAA) in the protein sample solution is 50 mM; the concentration of iodoacetamide (IAA) solution is 1 M; the amount of ammonium bicarbonate solution added is to reduce the urea concentration in the protein sample solution to below 1 M; the concentration of ammonium bicarbonate solution is 50 mM.

6. The use according to claim 2 or 5, characterized in that: In step (2), the time of being placed at room temperature is 3 hours; the time of being placed in a dark place at room temperature is 1 hour.

7. The use according to claim 2, characterized in that: In step (3), in the reduced alkylated protein solution, the mass ratio of protein to trypsin is 100:1; and the mass concentration of FA in the reduced alkylated protein solution is 0.5%.

8. The use according to claim 2 or 5, characterized in that: In step (3), the enzyme digestion is carried out at 37°C overnight; the desalting is carried out using Sep-Pak C 18 column.

9. The use according to any one of claims 2 to 8, characterized in that: In step (4), the liquid chromatography conditions are: Thermo Acclaim PepMap C 18 The desalting and enrichment were performed on a ThermoAcclaim PepMap C column (100 μm × 3.5 cm, 5 μm). 18 Separation on a column (75 μm×15 cm, 3 μm), mobile phase: 0.1% (v / v) formic acid in water (A) and 0.1% (v / v) formic acid in acetonitrile (B), gradient elution, flow rate: 300 nL·min -1 , injection volume: 1 µL; the gradient elution program was: 0~1 min, 99%→94%A; 1~96 min, 94%→78%A; 96~113 min, 78%→70%A; 113~117 min, 70%→5%A; 117~120 min, 5%A.

10. The use according to claim 9, characterized in that: The mass spectrometry conditions are as follows: analysis in positive ion mode, spray voltage: 2.1 kV, ion transmission capillary temperature: 275 °C, S-Lens transmission efficiency: 60%; primary mass spectrometry uses Orbitrap as a mass analyzer, with a resolution of 60000 and an acquisition range of 350-1550 ( m / z ); The secondary mass spectrometer also uses Orbitrap as the mass analyzer, the scanning mode is Data-Dependent MSn Scan mode, and the fragmentation mode is HCD mode.

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