A differential characteristic polypeptide library for identifying different medicinal parts of Bufo gargarizans and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a differential characteristic polypeptide library for identifying different medicinal parts of Chinese giant toads and its application. In the prior art, there are differences in protein components between toad venom and toad skin, toad meat, and toad bones, 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 perform identification and analysis on toad venom and dried toad medicinal materials, which has strong specificity, good stability, is not disturbed by the processing process, and can identify multiple medicinal materials at the same time, with obvious advantages.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a differential characteristic polypeptide library for identifying different medicinal parts of Bufo gargarizans and its application. Background Art

[0002] Bufo gargarizans Bufo gargarizans is a commonly used medicinal source animal in traditional Chinese medicine clinical practice. The toad is "pungent in taste, cool in nature, and poisonous. It belongs to the heart, liver, spleen, and lung meridians", and can "detoxify and disperse nodules, eliminate accumulation and promote diuresis, kill insects and relieve infantile malnutrition. It is mainly used to treat carbuncles, boils, sores on the back, scrofula, malignant sores, mass accumulation, distension, edema, infantile malnutrition, tetanus, and chronic cough". Ancient and modern empirical formulas have widely used it for removing toxins and reducing swelling, relieving pain and killing insects, strengthening the heart and promoting diuresis. Modern clinical practice has also used it for anti-tumor, analgesia, promoting diuresis and reducing swelling, and enhancing the body's immunity. The whole body of the toad can be used as medicine, and commonly used ones include bufotalin, toad skin, etc.

[0003] In recent years, due to the continuous expansion of the clinical application of bufotalin and its high price, there are common phenomena of mixed varieties, adulteration, and counterfeiting in commercial bufotalin. Traditional methods such as morphological identification, microscopic identification, and physicochemical identification cannot effectively distinguish bufotalin from toad skin, toad meat, and toad bone, 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 gargarizans.

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

[0006] The technical solution adopted by the present invention to achieve the above object is as follows:

[0007] The present invention provides a differential characteristic polypeptide library for identifying different medicinal parts of Bufo gargarizans, and the characteristic polypeptide library is composed of sequences such as SEQ ID NO.1-29.

[0008] The present invention also provides the application of the above differential characteristic polypeptide library in quickly distinguishing and identifying different medicinal parts of Bufo gargarizans, including the following steps:

[0009] (1) Sample preparation: Weigh the sample powder precisely, add a protein lysis solution, extract by ultrasonic treatment, take the supernatant after centrifugation to obtain a protein sample solution;

[0010] (2) Reduction alkylation: Take the protein sample solution, add a dithiothreitol (DTT) solution, and let it stand at room temperature for a period of time; then add an iodoacetamide (IAA) solution and let it stand in the dark at room temperature for a period of time; then add an ammonium bicarbonate solution to obtain a reduced alkylated protein solution;

[0011] (3) Protease digestion and desalting: Add trypsin to the reduced and alkylated protein solution and digest overnight; then add FA to terminate the digestion reaction and perform desalting.

[0012] (4) Identification is carried out by triple quadrupole liquid chromatography-mass spectrometry.

[0013] Further, in step (1), the ratio of the sample powder to the protein lysis solution is 25 mg: 1 mL; the composition of the protein lysis solution is: 21.0 g of urea, 7.60 g of thiourea, 50 μL of Triton X-100, and make up the volume to 50 mL with 50 mM ammonium bicarbonate solution.

[0014] Further, in step (1), the time of ultrasonic extraction is 30 min; the centrifugation is carried out at 4 °C at 13000 r∙min -1 Centrifuge for 15 min.

[0015] Further, in step (2), the concentration of dithiothreitol (DTT) in the protein sample solution is 10 mM; the concentration of the dithiothreitol (DTT) solution is 1 M; the concentration of iodoacetamide (IAA) in the protein sample solution is 50 mM; the concentration of the iodoacetamide (IAA) solution is 1 M; the addition amount of the ammonium bicarbonate solution is to reduce the urea concentration in the protein sample solution to below 1 M; the concentration of the ammonium bicarbonate solution is 50 mM.

[0016] Further, in step (2), the time of standing at room temperature is 3 h; the time of standing in the dark at room temperature is 1 h.

[0017] Further, in step (3), in the reduced and alkylated protein solution, the mass ratio of the protein amount to trypsin is 100:1; the mass concentration of FA in the reduced and alkylated protein solution is 0.5%.

[0018] Further, in step (3), the digestion is carried out overnight at 37 °C; the desalting is carried out using a Sep-PakC 18 column.

[0019] Further, in step (4), the conditions of the liquid chromatography are: desalting and enrichment with a Thermo Acclaim PepMap C 18 column (100 μm×3.5 cm, 5 μm), and on a Thermo Acclaim PepMap C 18Separation was performed on a column (75 μm×15 cm, 3 μm), mobile phase: 0.1% (v / v) formic acid aqueous solution (A) and 0.1% (v / v) formic acid acetonitrile solution (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.

[0020] Furthermore, the conditions of the mass spectrometry were: analysis in positive ion mode, spray voltage: 2.1 kV, ion transfer capillary temperature: 275 °C, S-Lens transmission efficiency: 60%; Orbitrap was used as the mass analyzer for the first-order mass spectrometry, with a resolution of 60000 and a collection range of 350 - 1550 ( m / z ); Orbitrap was also used as the mass analyzer for the second-order mass spectrometry, scan mode: Data-Dependent MSn Scan mode, fragmentation mode: HCD mode.

[0021] In the prior art, there are differences in protein components among bufotoxin, dried toad skin, toad meat, and toad bone. However, the detection of protein macromolecules is relatively cumbersome, which is not conducive to the transformation of the method into a standard. The present invention uses polypeptides as index components to identify and analyze bufotoxin and dried toad medicinal materials, with strong specificity, good stability, not interfered by the processing process, and can identify multiple medicinal materials simultaneously, showing obvious advantages.

[0022] The beneficial effects of the present invention are as follows:

[0023] (1) The identification method provided by the present invention can simultaneously identify bufotoxin, dried toad skin, toad meat, and toad bone, with good specificity and high efficiency;

[0024] (2) The characteristic polypeptide library provided by the present invention has strong specificity. During the subsequent application process, the detection method is simple and fast. The establishment of a quantitative determination method for bufotoxin and an inspection method for the components of dried toad in bufotoxin provides method support for the quality control of bufotoxin and improves the accuracy of detection at the same time. Description of the Drawings

[0025] Figure 1 BSA standard curve;

[0026] Figure 2 Principal component analysis diagram of toad-derived related medicinal materials;

[0027] Figure 3 OPLS-DA diagram (A) and score diagram (B) for comparison between bufotoxin and dried toad skin;

[0028] Figure 4 OPLS-DA and score plots (A and B) for comparison between toad venom and toad meat;

[0029] Figure 5 OPLS-DA and score plots (A and B) for comparison between toad venom and toad bone;

[0030] Figure 6 Verification process of polypeptide sequence NNAYDINEER (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: Verification result by AB SCIEX 6500 ＋ liquid chromatography-mass spectrometry instrument)

[0031] Figure 7 Verification process of polypeptide sequence FLEYGHR (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: Verification result by AB SCIEX 6500 ＋ liquid chromatography-mass spectrometry instrument)

[0032] Figure 8 Verification process of polypeptide sequence NVWSPDEK (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: Verification result by AB SCIEX 6500 ＋ liquid chromatography-mass spectrometry instrument)

[0033] Figure 9 Verification process of polypeptide sequence KGQDFEVR (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: Verification result by AB SCIEX 6500 ＋ liquid chromatography-mass spectrometry instrument)

[0034] Figure 10 Verification process of polypeptide sequence FNEGHVLF (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: Verification result by AB SCIEX 6500 ＋ liquid chromatography-mass spectrometry instrument)

[0035] Figure 11 Verification process of polypeptide sequence SAWPLLPGK (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: Verification result by AB SCIEX 6500 ＋ liquid chromatography-mass spectrometry instrument)

[0036] Figure 12 Verification process of polypeptide sequence ASEPAGPADEVK (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: Verification result by AB SCIEX 6500＋ Verification Results of Liquid Chromatography-Mass Spectrometry

[0037] Figure 13 Verification Process of Polypeptide Sequence SAWPLF(sub L)PGK (A: Prediction Results of Peaks Studio Software, B: Results of Homology Analysis, C: AB SCIEX 6500 ＋ Verification Results of Liquid Chromatography-Mass Spectrometry, D: Verification Results of Nano LC-MS / MS);

[0038] Figure 14 Verification Process of Polypeptide Sequence GVAGPPGAVGASGK (A: Prediction Results of Peaks Studio Software, B: Results of Homology Analysis, C: AB SCIEX 6500 ＋ Verification Results of Liquid Chromatography-Mass Spectrometry)

[0039] Figure 15 Verification Process of Polypeptide Sequence GTVGLPGQR (A: Prediction Results of Peaks Studio Software, B: Results of Homology Analysis, C: AB SCIEX 6500 ＋ Verification Results of Liquid Chromatography-Mass Spectrometry)

[0040] Figure 16 Verification Process of Polypeptide Sequence ISNIPDEYFQGFK (A: Prediction Results of Peaks Studio Software, B: Results of Homology Analysis, C: AB SCIEX 6500 ＋ Verification Results of Liquid Chromatography-Mass Spectrometry)

[0041] Figure 17 Verification Process of Polypeptide Sequence GEIGPAGS(sub N)TGPTGAAGGR (A: Prediction Results of Peaks Studio Software, B: Results of Homology Analysis, C: AB SCIEX 6500 ＋ Verification Results of Liquid Chromatography-Mass Spectrometry, D: Verification Results of Nano LC-MS / MS);

[0042] Figure 18 Verification Process of Polypeptide Sequence SGEPGAAGPP(+15.99)GPP(+15.99)GEK (A: Prediction Results of Peaks Studio Software, B: Results of Homology Analysis, C: AB SCIEX 6500 + Verification Results of Liquid Chromatography-Mass Spectrometry, D: Verification Results of Nano LC-MS / MS);

[0043] Figure 19Verification process of polypeptide sequence GFPGLP(+15.99)GPNGEPGK (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: Verification result by AB SCIEX 6500 ＋ Liquid chromatography - mass spectrometry verification result, D: Nano LC - MS / MS verification result);

[0044] Figure 20 Verification process of polypeptide sequence GESGPAGPP(+15.99)GAPGAPGAP(+15.99)GPAGPAGK (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: AB SCIEX 6500 ＋ Liquid chromatography - mass spectrometry verification result, D: Nano LC - MS / MS verification result);

[0045] Figure 21 Verification process of polypeptide sequence QLFEGLSR (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: AB SCIEX 6500 ＋ Liquid chromatography - mass spectrometry verification result)

[0046] Figure 22 Verification process of polypeptide sequence LLAGQSIISSAR (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: AB SCIEX 6500 ＋ Liquid chromatography - mass spectrometry verification result)

[0047] Figure 23 Verification process of polypeptide sequence YEVTTLR (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: AB SCIEX 6500 ＋ Liquid chromatography - mass spectrometry verification result)

[0048] Figure 24 Verification process of polypeptide sequence DLEEATLQHEATAS(sub A)ALR (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: AB SCIEX 6500 ＋ Liquid chromatography - mass spectrometry verification result, D: Nano LC - MS / MS verification result);

[0049] Figure 25 Verification process of polypeptide sequence VTFQLPA(sub S)ER (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: AB SCIEX 6500 ＋Verification results of liquid chromatography-mass spectrometry (D: Verification results of Nano LC-MS / MS);

[0050] Figure 26 Verification process of polypeptide sequence SEEELAEC(+57.02)FR (A: Prediction results of Peaks Studio software, B: Homology analysis results, C: AB SCIEX 6500 ＋ Verification results of liquid chromatography-mass spectrometry (D: Verification results of Nano LC-MS / MS);

[0051] Figure 27 Verification process of polypeptide sequence VDMTTLR (A: Prediction results of Peaks Studio software, B: Homology analysis results, C: AB SCIEX 6500 ＋ Verification results of liquid chromatography-mass spectrometry

[0052] Figure 28 Verification process of polypeptide sequence VGLLNYASSVK (A: Prediction results of Peaks Studio software, B: Homology analysis results, C: AB SCIEX 6500 ＋ Verification results of liquid chromatography-mass spectrometry

[0053] Figure 29 Verification process of polypeptide sequence YPIVNPR (A: Prediction results of Peaks Studio software, B: Homology analysis results, C: AB SCIEX 6500 ＋ Verification results of liquid chromatography-mass spectrometry

[0054] Figure 30 Verification process of polypeptide sequence AGWLSDGSVR (A: Prediction results of Peaks Studio software, B: Homology analysis results, C: AB SCIEX 6500 ＋ Verification results of liquid chromatography-mass spectrometry

[0055] Figure 31 Verification process of polypeptide sequence YPIVTPR (A: Prediction results of Peaks Studio software, B: Homology analysis results, C: AB SCIEX 6500 ＋ Verification results of liquid chromatography-mass spectrometry

[0056] Figure 32 Verification process of polypeptide sequence GLAGPQGPR (A: Prediction results of Peaks Studio software, B: Homology analysis results, C: AB SCIEX 6500 ＋ Verification results of liquid chromatography-mass spectrometry

[0057] Figure 33 Verification process of polypeptide sequence TPQAFVC(+57.02)LPR (A: Prediction result by Peaks Studio software, B: Homology analysis result, C: Verification result by AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification result, D: Nano LC-MS / MS verification result);

[0058] Figure 34 Verification process of polypeptide sequence ETWVDAENLC(+57.02)R A: Prediction result by Peaks Studio software, B: Homology analysis result, C: AB SCIEX 6500 ＋ Liquid chromatography-mass spectrometry verification result, D: Nano LC-MS / MS verification result);

[0059] Figure 35 Typical diagram for toad venom identification result (taking the ion pair 461.2→661.3 as an example);

[0060] Figure 36 Typical diagram for toad skin screening result (taking the ion pair 718.8→867.4 as an example);

[0061] Figure 37 Typical diagram for toad meat screening result (taking the ion pair 619.3→818.4 as an example);

[0062] Figure 38 Typical diagram for toad bone determination result (taking the ion pair 418.2→612.4 as an example). Specific implementation manners

[0063] The technical solutions of the present invention will be further explained and illustrated through specific embodiments below.

[0064] The sample information used in the present invention is shown in Table 1.

[0065] Table 1 Sample information

[0066]

[0067] Analysis software: SIEVE 2.2, SIMCA 14.1 (32-bit).

[0068] Example 1

[0069] 1.1 Experimental method

[0070] 1.1.1 Sample preparation

[0071] Take about 250 mg of the sample powder, accurately weigh it, add 10 mL of protein lysis solution (21.0 g of urea, 7.60 g of thiourea, 50 μL of Triton X-100, and make up to 50 mL with 50 mM ammonium bicarbonate solution), extract by ultrasound for 30 min, let stand overnight, centrifuge at 4 °C for 15 min (13000 r∙min -1 ), take the supernatant to obtain the protein sample solution for subsequent experiments.

[0072] 1.1.2 Protein concentration determination

[0073] Use a modified Bradford method protein concentration assay kit to determine the protein concentration.

[0074] Preparation of standard solution: Pipette 0, 50, 100, 150, 200, 250, 300 μL of 1.00 mg·mL -1 BSA standard protein into 2 mL centrifuge tubes respectively, and then add 1000, 950, 900, 850, 800, 750, 700 μL of pure water for dilution respectively to obtain BSA standard protein solutions with concentrations of 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30 mg·mL -1 respectively.

[0075] Precisely pipette 20 μL of BSA standard protein and toad venom protein solution with different concentrations into different wells of a 96-well plate respectively, then add 200 μL of Bradford working solution into the wells, and measure the absorbance value at 595 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0076] 1.1.3 Reduction alkylation

[0077] According to the protein solution concentration determination result, take 250 μg of the protein sample, add an appropriate amount of 1 M dithiothreitol (DTT) solution to make the final concentration of DTT 10 mM, and let stand at room temperature for 3 h. Then add an appropriate amount of 1 M iodoacetamide (IAA) solution to make the final concentration of IAA 50 mM, and let stand at room temperature in the dark for 1 h. Then add 50 mM ammonium bicarbonate solution to reduce the urea concentration to less than 1 M.

[0078] 1.1.4 Protease digestion and desalting

[0079] According to the ratio of protein amount: trypsin amount = 100:1, add trypsin, and digest in a constant temperature and humidity box at 37 °C overnight (12 h). The next day, add an appropriate amount of FA to make the final concentration of FA 0.5% (mass concentration) to terminate the digestion reaction. Use a Sep-Pak C 18 column for desalting.

[0080] 1.1.5 Nano analysis conditions

[0081] Chromatographic conditions: Desalting and enrichment were performed using a Thermo Acclaim PepMap C 18 column (100 μm×3.5 cm, 5 μm), and separation was carried out on a Thermo Acclaim PepMap C 18 column (75 μm×15 cm, 3 μm). Mobile phase: 0.1% (v / v) formic acid aqueous solution (A) and 0.1% (v / v) formic acid acetonitrile solution (B), 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), flow rate: 300 nL·min -1 , injection volume: 1 μL.

[0082] Mass spectrometry conditions: Analysis was performed in positive ion mode, spray voltage: 2.1 kV, ion transfer capillary temperature: 275 °C, S-Lens transmission efficiency: 60%; Orbitrap was used as the mass analyzer for the first-stage mass spectrometry, with a resolution of 60000 and a collection range of 350 - 1550 ( m / z ); Orbitrap was also used as the mass analyzer for the second-stage mass spectrometry, scanning mode: Data-Dependent MSn Scan mode, fragmentation mode: HCD mode.

[0083] 1.1.6 Peaks database search

[0084] Peaks Studio software (version 8.5) was used to identify the peak components in combination with the Bufo gargarizans database downloaded from NCBI. The parameter settings were as follows: Trypsin was selected, with a maximum of 6 missed cleavages. Oxidation (+15.99), hydroxylation (+15.99), deamidation (+0.98), and acetylation of the protein N-terminus (+42.01) were specified as variable modifications. The maximum parent ion tolerance was 15 ppm, the fragment ion tolerance was 0.02 Da, the filtered charge was set to 2 - 8, and all other parameters were default settings. The FDR (false discovery rate) for polypeptides and proteins was set to 1%.

[0085] 1.1.7 Chemometric analysis

[0086] Based on the polypeptide spectra, SIEVE and SIMCA software were used to screen for mass spectrometry characteristic ions and find the mass spectrometry information for differentiating chan su from chan pi, chan meat, and chan bone.

[0087] 1.1.7.1 Pretreatment of mass spectrometry data

[0088] The data collected by high-resolution mass spectrometry was imported into SIEVE 2.2 software. The toad venom samples were selected as reference samples, and all samples were subjected to peak alignment and framing. Information such as the accurate mass-to-charge ratio, retention time, and peak area of the ions was extracted, and each ion pair was numbered for subsequent chemometric analysis.

[0089] 1.1.8 Specificity verification method

[0090] The candidate potential characteristic ions must be verified by mass spectrometry for their actual presence in different toad venom, toad skin, toad meat, and toad bone samples to determine their exclusivity. Using the multiple reaction monitoring mode (MRM) of the AB SCIEX 6500 ＋ triple quadrupole mass spectrometer, verification was carried out through chromatographic behavior. According to the presence or absence and response of the chromatographic peaks of the potential characteristic ions in different toad venom, toad skin, toad meat, and toad bone samples, the exclusivity of the characteristic ions was confirmed.

[0091] Chromatographic method: The sample was chromatographically separated using an Agilent Eclipse C 18 column (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 temperature was kept at 43 °C. The mobile phase consisted of A (0.1% formic acid aqueous solution) 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.

[0092] Mass spectrometry method: The LC-MS / MS system was controlled using Analyst Software. The parameter settings were as follows: The mode was set to electrospray ionization (ESI) and positive ion MRM mode. Sheath Gas flow rate: 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.

[0093] Example 2 Result analysis

[0094] 1.1 Chemometric results

[0095] Figure 2It 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.

[0096] 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.

[0097] 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.

[0098] 2.1 Experimental Materials

[0099] During the detection process, some biomarkers were synthesized, and the detailed information is shown in Table 2.

[0100] Table 2 Information table of peptide biomarker reference substances

[0101]

[0102] 2.2 Experimental methods

[0103] 2.2.1 Sample preparation

[0104] 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.

[0105] Preparation of spiked samples: According to the response values ​​of synthetic peptides and samples, the two were mixed in proportion to prepare spiked samples.

[0106] The reference stock solution, test solution and spiked solution were diluted to appropriate concentrations and injected for analysis.

[0107] 2.2.2 Analysis conditions

[0108] AB SCIEX 6500 ＋ The chromatographic and mass spectrometric conditions of the liquid chromatography-mass spectrometer are the same as those in Example 1.

[0109] 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.

[0110] 2.3 Experimental Results

[0111] There are 7 differential polypeptide sequences in chan su without modification and 1 differential polypeptide sequence with modification, specifically as Figure 6 shown

[0112] There are 3 differential polypeptide sequences in chan pi without modification and 4 differential polypeptide sequences with modification. The confirmed sequences are as Figures 7 - 10 shown

[0113] There are 3 differential polypeptide sequences in chan rou without modification and 3 differential polypeptide sequences with modification. The confirmed sequences are as Figures 11 - 13 shown

[0114] There are 6 differential polypeptide sequences in chan gu without modification and 2 differential polypeptide sequences with modification. The confirmed sequences are as Figure 14 and Figure 15 shown

[0115] Example 3

[0116] 1 Sample Preparation

[0117] According to the prescription amount of chan su in the preparations (Xiongdan Jiuxin Pills, Liuling Pills, Houzheng Pills, Niuhuang Xiaoyan Tablets), weigh an equivalent amount of the preparations, and prepare the sample solution according to the sample preparation method in the 1.1 experimental method in Example 1

[0118] 2 Analytical Conditions

[0119] Chromatographic method: Chromatographic 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 aqueous solution (A) and acetonitrile solution (B), gradient elution 0 - 20 min, 3 - 25%B, 20 - 21 min, 25 - 90%B. Injection volume 5 µL

[0120] Mass spectrometry method: Electrospray ionization source (ESI), MRM monitoring in positive ion mode: scanning range m / z : 100 - 1250; IS: 5500 V, TEM: 550 °C; CUR: 30 psi; GS1: 50 psi; GS2: 50 psi; CAD: 8

[0121] 3 Experimental Results

[0122] Chan su polypeptide signals are detected in all four preparations, and at the same time, chan pi components are also detected. Chan rou polypeptide signals are detected in Xiongdan Jiuxin Pills and Niuhuang Xiaoyan Tablets. The results are as Figures 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 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 the protein sample solution, add dithiothreitol solution, and leave it at room temperature for a period of time; Then add iodoacetamide solution and place in dark place at room temperature for a while; then add ammonium bicarbonate solution to obtain reduced alkylated protein solution; (3) Enzyme cleavage and desalting: Add trypsin to the reduced alkylated protein solution and digest overnight; then add formic acid 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 in the protein sample solution is 10 mM; the concentration of the dithiothreitol solution is 1 M; the concentration of iodoacetamide in the protein sample solution is 50 mM; the concentration of the iodoacetamide 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 the 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 formic acid 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 claim 2, characterized in that: In step (4), the liquid chromatography conditions are: Thermo Acclaim PepMap C 18 Column, 100 μm × 3.5 cm, 5 μm, desalting and enrichment, 75 μm × 15 cm, 3 μm Thermo Acclaim PepMap C 18 On-column separation, mobile phase: A is 0.1%, v / v formic acid in water and B is 0.1%, v / v formic acid in acetonitrile, 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; 113~117min, 70%→5%A; 117~120 min, 5%A.

10. The use according to claim 9, characterized in that: The mass spectrometry conditions were 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 used Orbitrap as a mass analyzer, with a resolution of 60,000 and an acquisition range of 350-1550 m / z ; The secondary mass spectrometry also used Orbitrap as the mass analyzer, scanning mode: Data-Dependent MSn Scan mode, fragmentation mode: HCD mode.

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