CN115184480A - Characteristic polypeptide for identifying cinobufotalin preparation or toad skin and content determination method and application thereof - Google Patents
Characteristic polypeptide for identifying cinobufotalin preparation or toad skin and content determination method and application thereof Download PDFInfo
- Publication number
- CN115184480A CN115184480A CN202210569411.7A CN202210569411A CN115184480A CN 115184480 A CN115184480 A CN 115184480A CN 202210569411 A CN202210569411 A CN 202210569411A CN 115184480 A CN115184480 A CN 115184480A
- Authority
- CN
- China
- Prior art keywords
- preparation
- pep
- cinobufagin
- polypeptide
- toad skin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 81
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 56
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 47
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 29
- KBKUJJFDSHBPPA-UHFFFAOYSA-N 5beta-hydroxylcinobufagin Natural products CC(=O)OC1C2OC22C3CCC4(O)CC(O)CCC4(C)C3CCC2(C)C1C=1C=CC(=O)OC=1 KBKUJJFDSHBPPA-UHFFFAOYSA-N 0.000 title claims description 21
- KBKUJJFDSHBPPA-ZNCGZLKOSA-N cinobufotalin Chemical compound C=1([C@@H]2[C@@]3(C)CC[C@@H]4[C@@]5(C)CC[C@H](O)C[C@@]5(O)CC[C@H]4[C@@]43O[C@@H]4[C@@H]2OC(=O)C)C=CC(=O)OC=1 KBKUJJFDSHBPPA-ZNCGZLKOSA-N 0.000 title claims description 21
- SCULJPGYOQQXTK-OLRINKBESA-N Cinobufagin Chemical compound C=1([C@@H]2[C@@]3(C)CC[C@@H]4[C@@]5(C)CC[C@H](O)C[C@H]5CC[C@H]4[C@@]43O[C@@H]4[C@@H]2OC(=O)C)C=CC(=O)OC=1 SCULJPGYOQQXTK-OLRINKBESA-N 0.000 claims abstract description 57
- SCULJPGYOQQXTK-UHFFFAOYSA-N Cinobufagin Natural products CC(=O)OC1C2OC22C3CCC4CC(O)CCC4(C)C3CCC2(C)C1C=1C=CC(=O)OC=1 SCULJPGYOQQXTK-UHFFFAOYSA-N 0.000 claims abstract description 57
- 238000001819 mass spectrum Methods 0.000 claims abstract description 16
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 15
- 238000004458 analytical method Methods 0.000 claims abstract description 14
- 238000003908 quality control method Methods 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 42
- 108010033276 Peptide Fragments Proteins 0.000 claims description 29
- 102000007079 Peptide Fragments Human genes 0.000 claims description 29
- 238000002156 mixing Methods 0.000 claims description 21
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- 239000000523 sample Substances 0.000 claims description 20
- 239000012086 standard solution Substances 0.000 claims description 12
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- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 239000012085 test solution Substances 0.000 claims description 11
- 101100189913 Caenorhabditis elegans pept-1 gene Proteins 0.000 claims description 10
- 101100494726 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) pep-4 gene Proteins 0.000 claims description 10
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- 239000000126 substance Substances 0.000 claims description 7
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 5
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 claims description 5
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 4
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- 230000000694 effects Effects 0.000 abstract description 6
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 7
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- 238000002955 isolation Methods 0.000 description 4
- 108010024654 phenylalanyl-prolyl-alanine Proteins 0.000 description 4
- HAJGVUYNXHQLER-UHFFFAOYSA-N Bufadienolide Natural products O1C(=O)C=CC(C2C3C(C4C(C5CCCCC5CC4)CC3)CC2)=C1 HAJGVUYNXHQLER-UHFFFAOYSA-N 0.000 description 3
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- XMPXVJIDADUOQB-RCOVLWMOSA-N Gly-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C([O-])=O)NC(=O)CNC(=O)C[NH3+] XMPXVJIDADUOQB-RCOVLWMOSA-N 0.000 description 3
- UGTHTQWIQKEDEH-BQBZGAKWSA-N L-alanyl-L-prolylglycine zwitterion Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UGTHTQWIQKEDEH-BQBZGAKWSA-N 0.000 description 3
- DCRWPTBMWMGADO-AVGNSLFASA-N Lys-Glu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O DCRWPTBMWMGADO-AVGNSLFASA-N 0.000 description 3
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 3
- 108010005233 alanylglutamic acid Proteins 0.000 description 3
- YBPMPRDOWHIVNA-XTBIJCDISA-N bufadienolide Chemical compound C=1([C@H]2CC[C@@H]3[C@H]4[C@@H]([C@]5(CCCCC5CC4)C)CC[C@@]32C)C=CC(=O)OC=1 YBPMPRDOWHIVNA-XTBIJCDISA-N 0.000 description 3
- PXOHOSHERMSUCD-RTBYDAEBSA-N bufanolide Chemical compound C([C@H]1[C@H]2[C@@H]([C@]3(CCCCC3CC2)C)CC[C@@]11C)CC1[C@H]1CCC(=O)OC1 PXOHOSHERMSUCD-RTBYDAEBSA-N 0.000 description 3
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- KIUYPHAMDKDICO-WHFBIAKZSA-N Ala-Asp-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KIUYPHAMDKDICO-WHFBIAKZSA-N 0.000 description 2
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- CRZRTKAVUUGKEQ-ACZMJKKPSA-N Ser-Gln-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O CRZRTKAVUUGKEQ-ACZMJKKPSA-N 0.000 description 1
- VDVYTKZBMFADQH-AVGNSLFASA-N Ser-Gln-Tyr Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 VDVYTKZBMFADQH-AVGNSLFASA-N 0.000 description 1
- SNVIOQXAHVORQM-WDSKDSINSA-N Ser-Gly-Gln Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O SNVIOQXAHVORQM-WDSKDSINSA-N 0.000 description 1
- GJFYFGOEWLDQGW-GUBZILKMSA-N Ser-Leu-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CO)N GJFYFGOEWLDQGW-GUBZILKMSA-N 0.000 description 1
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 1
- ASGYVPAVFNDZMA-GUBZILKMSA-N Ser-Met-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CO)N ASGYVPAVFNDZMA-GUBZILKMSA-N 0.000 description 1
- AABIBDJHSKIMJK-FXQIFTODSA-N Ser-Ser-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(O)=O AABIBDJHSKIMJK-FXQIFTODSA-N 0.000 description 1
- ILZAUMFXKSIUEF-SRVKXCTJSA-N Ser-Ser-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ILZAUMFXKSIUEF-SRVKXCTJSA-N 0.000 description 1
- PYTKULIABVRXSC-BWBBJGPYSA-N Ser-Ser-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PYTKULIABVRXSC-BWBBJGPYSA-N 0.000 description 1
- PCJLFYBAQZQOFE-KATARQTJSA-N Ser-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N)O PCJLFYBAQZQOFE-KATARQTJSA-N 0.000 description 1
- ZKOKTQPHFMRSJP-YJRXYDGGSA-N Ser-Thr-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZKOKTQPHFMRSJP-YJRXYDGGSA-N 0.000 description 1
- JWQNAFHCXKVZKZ-UVOCVTCTSA-N Thr-Lys-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JWQNAFHCXKVZKZ-UVOCVTCTSA-N 0.000 description 1
- DOBIBIXIHJKVJF-XKBZYTNZSA-N Thr-Ser-Gln Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O DOBIBIXIHJKVJF-XKBZYTNZSA-N 0.000 description 1
- LVRFMARKDGGZMX-IZPVPAKOSA-N Thr-Tyr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)O)C(O)=O)CC1=CC=C(O)C=C1 LVRFMARKDGGZMX-IZPVPAKOSA-N 0.000 description 1
- UDLYXGYWTVOIKU-QXEWZRGKSA-N Val-Asn-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N UDLYXGYWTVOIKU-QXEWZRGKSA-N 0.000 description 1
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Abstract
The invention discloses a characteristic polypeptide for identifying cinobufagin preparation or toad skin, a content determination method and application thereof. The invention identifies the polypeptide in the cinobufagin preparation by a proteomics analysis means based on mass spectrum, establishes a quantitative method of the polypeptide in the cinobufagin preparation by a mass spectrum targeted proteomics analysis technology, finds out the key characteristic polypeptide components of the cinobufagin preparation and the raw material toad skin thereof, provides key indexes for realizing the comprehensive quality analysis and quality control of the cinobufagin preparation, and ensures the quality and clinical curative effect of the cinobufagin preparation.
Description
Technical Field
The invention relates to a characteristic polypeptide for identifying cinobufagin preparation or toad skin, and a content determination method and application thereof, and belongs to the technical field of biological medicine.
Background
The skin of Bufo Bufo gargarizans Cantor or Bufo melanostictus Schneider, which is the animal of Bufo Bufo gargarizans Cantor, has the effects of clearing away heat and toxic materials, inducing diuresis and relieving edema. In traditional Chinese medicine diagnosis and treatment, the toad skin can be used for treating symptoms such as casing examination secret, carbuncle sore examination secret and intestine head emergence examination secret. Cinobufotalin is a traditional Chinese medicine preparation, is prepared by processing sterilized water extract of dried skin of Bufo bufo gargarizans Cantor, and has the effects of detoxifying, relieving swelling and relieving pain. Cinobufotalin contains polypeptide, nucleic acid, indole alkaloids, and bufanolide. In clinical treatment, cinobufagin can be used for resisting inflammation and easing pain, treating middle and late stage tumors, chronic hepatitis B and other symptoms, and is a clinically common anti-tumor drug. In the treatment of tumor, the medicament is often combined with cisplatin and other therapeutic drugs, so that the toxic and side effects of chemotherapy are reduced, and the treatment effectiveness is improved. In recent years, the research on cinobufagin focuses on small molecular indole alkaloids and bufanolide in cinobufagin. Wherein the bufanolide (bufadienolide) component is considered as the main active substance in cinobufagin. However, although the bufadienolide has a remarkable curative effect, the treatment window is narrow, and the bufadienolide has remarkable cardiotoxicity. The toad skin and cinobufotalin have high polypeptide component content, no cardiac toxicity and various pharmacological activities, and are key components of cinobufotalin preparations. But methods of identification and quality control have long been lacking.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to overcome the defects of the prior art and provides a quality control method for characteristic peptide fragments and liquid phase-mass spectrometry combined analysis in cinobufagin preparation and toad skin.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the technical scheme that:
a characteristic polypeptide for identifying cinobufagin preparation or toad skin, wherein the characteristic polypeptide comprises one or more of PEP 1-PEP 25; the PEP 1-PEP 25 sequence is AVFPSIVGRPR, PEPAAKSAPAPK, GIADALGKAYH, GVTIAQGGVLPNIQ, AP (+ 15.99) GQPQLQISGQ, APGP (+ 15.99) KGERGL, A (+ 42.01) STKEKLIHN, PPPGGPYPGP, AGEKVIKETGGIIGGIGDAIGGLGK, PIFPAAPASM (+ 15.99) GFN, E SVG (+ 42.01) EEIAVQLVDNGSGM (+ 15.99), PVPYWEVQPATFR, A (+ 42.01) SLQQVNQQQQQQQQQQQFTLINLKAN, PTFPALQKKHDISHDISSQR, S (+ 42.01) GFSTKTASTGQPN, S GQSAVQPSK, PVIQWGIQGRS, PVGGKEPNPSTP, AGFAQASSSGRESSR, PVAGFAQASTGASTGS (+ 15.01) GAAGASAGGRAGGRAGGRASQASTGS (+ 15.15.15.15.15.15.15 ASGGASAGGRASAGGRASIGGGASTGS.
AVFPSIVGRPR is Ala Val Phe Pro Ser Ile Val Gly Arg Pro Arg;
PEPAAKSAPAPK is Pro Glu Pro Ala Lys Ser Ala Pro Ala Pro Lys;
GIADALGKAYH is Gly Ile Ala Asp Ala Leu Gly Lys Ala Tyr His;
GVTIAQGGVLPNIQ is Gly Val Thr Ile Ala Gln Gly Val Leu Pro Asn Ile Gln;
AP (+ 15.99) GQPQLQISGQ is Ala Pro (+ 15.99) Gly Gln Pro Gln Leu Gln Ile Ser Gly Gln;
APGP (+ 15.99) KGERGL is Ala Pro Gly Pro (+ 15.99) Lys Gly Glu Arg Gly Leu;
a (+ 42.01) STKEKLIHN is Ala (+ 42.01) Ser Thr Lys Glu Lys Leu Ile His Asn;
PPPGPGGPYPGP is Pro Pro Gly Pro Gly Gly Pro Gly Tyr Pro Gly Pro;
AGEKVIKETGGIIHGIGDAIGGLGK is Ala Gly Glu Lys Val Ile Lys Glu Thr Gly Gly Ile Ile His Gly Ile Gly Asp Ala Ile Gly Leu Gly Lys;
PIFPAAPASM (+ 15.99) GFN is Pro Ile Phe Pro Ala Ala Pro Ala Ser Met (+ 15.99) Gly Phe Asn;
e (+ 42.01) EEIAALVVDNGSGM (+ 15.99) Glu (+ 42.01) Glu Ile Ala Ala Leu Val Asp Asn Gly Ser Gly Met (+ 15.99);
PVPYWEVQPATFR is Pro Val Pro Tyr Trp Glu Val Gln Pro Ala Thr Phe Arg;
a (+ 42.01) SLQLINVNFQQ is Ala (+ 42.01) Ser Leu Gln Leu Ile Asn Val Asn Phe Gln Gln;
PTTEFLLKAN is Pro Thr Thr Glu Phe Leu Leu Lys Ala Asn;
the PTFPALTAQKKELHDIAQR is Pro Thr Glu Phe Pro Ala Leu Thr Ala Glu Gln Lys Glu Leu His Asp Ile Ala Gln Arg;
s (+ 42.01) RQSTYTKTFSAASAVQPRPN is Ser (+ 42.01) Arg Gln Ser Thr Tyr Thr Lys Thr Phe Ser Ala Val Gln Pro Arg Pro Asn;
TLWGIQKELQF is Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe;
PVILGKYPNSFT is Pro Val Ile Leu Gly Lys Tyr Pro Asn Ser Phe Thr;
AGFAGDDAPRAVFPSIVGRPR is Ala Gly Phe Ala Gly Asp Ala Pro Arg Ala Val Phe Pro Ser Ile Val Gly Arg Pro Arg Ala;
ATGQAGPP (+ 15.99) GPVGP (+ 15.99) is Ala Thr Gly Gln Ala Gly Pro Pro (+ 15.99) Gly Pro Val Gly Pro (+ 15.99);
PVQYWEIQPATFR is Pro Val Gln Tyr Trp Glu Ile Gln Pro Ala Thr Phe Arg;
HIQEVAESVGTGGIMGK is His Ile Gln Glu Val Ala Glu Ser Val Gly Thr Gly Ile Met Gly Lys;
KPQDDSANAGGISSTSAVNR is Lys Pro Gln Asp Ser Ser Ala Asn Gly Ile Ser Thr Ser Ala Ser Val Asn Arg;
t (+ 42.01) SQYARSLGGGQSAGSSSFR is Thr (+ 42.01) Ser Gln Tyr Ala Arg Ser Leu Gly Gly Gln Ser Ala Gly Ser Ser Ser Ser Ser Phe Arg;
LDRLLEGGSLSSQASMVAGAG is Leu Asp Arg Leu Glu Gly Ser Leu Ser Gln Ala Ser Ser Met Val Ala Ser Gly Ala Asp Gly.
Further, the cinobufotalin preparation comprises at least one of a cinobufotalin capsule, a cinobufotalin injection and a cinobufotalin tablet. Specifically, the cinobufotalin preparation is derived from Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider.
A quantitative analysis method for identifying characteristic polypeptide of cinobufagin preparation or toad skin, comprising the following steps:
(1) Preparation of a standard solution: preparing mixed standard solution by taking polypeptides PEP 1-PEP 4, namely AVFPSIVGRPR, PEPAAKSAPAPK, GIADALGKAYH and GVTIAQGGVLPNIQ, and diluting to obtain a series of standard solution with the concentration range of 6.4 ng/mL-100 mu g/mL;
(2) Preparation of a test solution: extracting cinobufagin preparation polypeptide or toad skin test solution;
(3) Establishing a standard curve equation: injecting the series of standard substance solutions in the step (1) into a liquid chromatography-mass spectrometer for analysis, and drawing a standard curve equation by taking the peak area as a vertical coordinate and the concentration as a horizontal coordinate;
(4) Content determination: and (3) injecting the cinobufagin preparation polypeptide or toad skin test solution obtained in the step (2) into a liquid chromatography-mass spectrometer, and calculating the content of the peptide segments shown by PEP 1-PEP 4 according to the standard curve equation obtained in the step (3).
As a preferable scheme, in the above quantitative analysis method for identifying a polypeptide characteristic of a cinobufagin preparation or toad skin, the preparation method of the test solution in step (2) is:
taking a cinobufagin preparation sample, taking supernatant for ultrafiltration treatment, concentrating and drying ultrafiltration liquid, and preparing a cinobufagin preparation extracting solution with the concentration of 5 mg/mL-15 mg/mL; sequentially carrying out disulfide reduction and alkylation treatment on the extracting solution, volatilizing, and re-dissolving with formic acid-water to obtain a cinobufagin preparation test sample solution;
or weighing and shearing toad skin, adding ultrapure water, decocting and extracting, mixing decoction and extraction filtrates, concentrating, adding ethanol into the concentrated solution to adjust the ethanol content to be 60-70%, uniformly mixing, standing, filtering out supernatant, and recovering ethanol until no ethanol smell exists; adding ethanol again to adjust the alcohol content to 80-85%, mixing uniformly, standing, filtering out supernatant, and recovering ethanol until no alcohol smell exists, namely the toad skin polypeptide extract; adding a certain volume of toad skin polypeptide extract into a disulfide reducing agent respectively, incubating in a constant-temperature mixing instrument, adding an alkylation reagent, incubating in a constant-temperature mixing instrument in a dark place, desalting a sample by using a desalting column, combining and volatilizing, and finally re-dissolving with formic acid-water to obtain a toad skin test solution.
Preferably, the quantitative analysis method for identifying the polypeptide characteristic of the cinobufagin preparation or the toad skin is used, and the disulfide reducing agent comprises dithiothreitol; the alkylating agent includes iodoacetamide.
Preferably, in the quantitative analysis method for identifying characteristic polypeptides of cinobufagin preparation or toad skin, the chromatographic conditions of the liquid chromatography-mass spectrometer analysis in the steps (3) and (4) are as follows: adopting a polar group bonded C18 chromatographic column, carrying out gradient elution by using 0.05-0.2% formic acid-water solution as a mobile phase A and 0.05-0.2% formic acid-acetonitrile solution as a mobile phase B at the flow rate of 0.1-0.5 mL/min, wherein the elution procedure is as follows: 0-1min, 60-90% by weight of B,1-1.5min, 90-90% by weight of B,1.5-2min, 90-60% by weight of B; sample injection amount: 2 mu L of the solution; flow rate: 0.3mL/min, column temperature: 30-50 ℃;
the mass spectrum conditions are as follows: ESI positive ionization mode and SRM scanning mode are adopted; the source parameters of mass spectrum detection ESI positive ionization mode are as follows: the spraying voltage is 3500V, the evaporation temperature is 300 ℃, the sheath gas pressure is 45psi, the ion scanning air pressure is 2.0psi, the auxiliary gas pressure is 15psi, and the capillary temperature is 270 ℃.
Further, in the step (3), online optimization and determination of mass spectrum parameters are also included: constructing three pairs of ion pairs by using the single-charge ion fragments and the double/triple-charge excimer ions of the peptide fragments as parent ions, generating different relative response values by using different collision energies, constructing a relative response value-collision energy curve of the candidate ion pairs by using Gaussian curve fitting, and taking a CE value corresponding to the highest point of the curve as an optimal CE value; different parent ions correspond to different ion introduction ring projection frequency amplitudes (S-Lens RF), a candidate parent ion relative response value-projection frequency amplitude curve is constructed by Gaussian curve fitting, and the S-Lens RF value corresponding to the highest point of the curve is used as an optimal S-Lens RF value; and detecting according to the optimized mass spectrum parameters.
Preferably, the method for the quantitative analysis of characteristic polypeptides for identifying cinobufagin preparations or toad skin is characterized by comprising the following steps: the standard curve equation and the linear range established in the step (3) are as follows:
| PEP No. | peptide fragment sequence | Regression equation | R 2 | Linear range |
| PEP 1 | AVFPSIVGRPR | y＝217.318x+5513.62 | 0.9985 | 6.4ng/mL～4μg/mL |
| PEP 2 | PEPAKSAPAPK | y＝326.786x-35487.9 | 0.9968 | 12.8ng/mL～4μg/mL |
| PEP 3 | GIADALGKAYH | y＝1182.24x-56961.1 | 0.9990 | 6.4ng/mL～4μg/mL |
| PEP 4 | GVTIAQGGVLPNIQ | y＝3547.23x-10284.5 | 0.9901 | 0.32μg/mL～100μg/mL |
In the cinobufotalin preparation and the raw material toad skin, the contents of the characteristic peptide segments are different, so that the blank of component research of the cinobufotalin preparation can be filled, and a key index is provided for realizing comprehensive quality analysis and quality control of the cinobufotalin preparation. Kits for preparing the characteristic peptide fragments can be prepared.
The beneficial effects are as follows:
the invention can sensitively detect the peptide fragment information through the targeted polypeptide omics analysis technology and has high repeatability. The multiple reaction monitoring mode (MRM mode) is a targeted proteomics quantification technique based on low resolution mass spectrometry. The target peptide fragment is targeted by the selected parent ions and the selected daughter ions, and the target peptide fragment can be quantified by establishing a standard curve. The target quantification method based on the parent ions and the daughter ions has the characteristics of high sensitivity and good reproducibility, and can simultaneously detect various substances by setting different ion pairs, so that the detection efficiency is improved. The invention establishes a quantitative method of characteristic peptide segments in the cinobufagin preparation by using mass spectrometry proteomics quantitative technology, finds the content difference of the peptide segments between the finished product preparation and the raw material toad skin, can fill the blank in the research of the cinobufagin preparation, provides key indexes for realizing the comprehensive quality analysis and quality control of the cinobufagin preparation, and ensures the quality and clinical curative effect of the cinobufagin preparation.
Drawings
FIG. 1 is an LC-MS selective ion detection chromatogram for 4 peptide fragments in cinobufagin preparation in the embodiment of the present invention (A: mixed standard; B: cinobufagin preparation; C: toad skin).
FIG. 2 is a mass spectrum of peptide fragments PEP 1-PEP 4.
Detailed Description
The invention is further illustrated by the following figures and examples.
Example 1
1. Analytical selection of target peptide fragments
1. Preparation of cinobufotalin preparation sample
Taking 200mg of cinobufagin preparation sample, adding 5mL of ultrapure water for dissolving, uniformly mixing by vortex, ultrasonically extracting for 30min in ice-water bath, 14000r,20min, centrifuging at 4 ℃ and taking supernatant; the obtained residue was further subjected to ultrasonic extraction in an ice-water bath with 5ml of ultrapure water for 30min, 14000r,20min and centrifugation at 4 ℃ to obtain a supernatant. Mixing the supernatants, adding into 10KD ultrafiltration tube, centrifuging at 8000r,20min at 4 ℃, sucking lower layer sample, and repeatedly ultrafiltering to combine the lower layer solution as cinobufagin preparation sample.
2. Polypeptide sample preparation
Adding a certain volume of cinobufagin (the amount of polypeptide is 20-200 mug) into a certain volume of dithiothreitol (the final concentration is 5 mM) respectively, incubating for 1h at 37 ℃ in a constant-temperature mixing instrument, adding a certain volume of iodoacetamide (the final concentration is 0.15M), and incubating for 45min at 37 ℃ in a dark place. Desalting the sample with desalting column, mixing, volatilizing, and re-dissolving with 0.1% formic acid-water to obtain cinobufagin sample.
3. Qualitative analysis
The samples are injected into a Nano LC-QE-Orbitrap-MS system, and high-resolution mass spectrum data of the samples are collected. Using water (containing 0.1% formic acid) as a flow line A, acetonitrile (containing 0.1% formic acid) as a mobile phase B, and mixing the mobile phase A and the mobile phase B to obtain an AB mixed solution for gradient elution, wherein the elution procedure is as follows: 0-2min,2% -5%; 2-78min,5% -32% B; 78-83min,32% -45% B;83-85min,45% -100% B;85-90min,100% B.
The acquisition time of the atlas is 90min. The mass spectrum detection adopts an ESI positive ion scanning mode, the primary mass spectrum scanning range m/z is 300-1,700, the resolution is 70,000, the automatic gain control target (AGC target) is 3 × e6, and the Maximum residence time (Maximum IT) is 50ms. The secondary mass spectral resolution was 17,500, the target was automatically gain controlled at 1 × e5, the maximum residence time was 100ms, the Isolation width of the parent ion (Isolation window) was 2m/z, the dynamic exclusion was turned on, the time was 30s, and the HCD high-energy collision mode was used.
Analyzing the original data of the mass spectrum by using PEAKS Studio 8.5 software, setting the error range of the mass of the precursor protein and the fragment to be 10ppm and 0.1Da, and checking and removing the pollution protein. And N-terminal acetylation (+ 42.01), hydroxylation (+ 15.99), oxidation (+ 15.99), carbamoylmethylation (+ 57.02), deamidation (+ 0.98) were set as fixed modifications. Database selection transcriptome self-establishes a database. The obtained high-response peptide fragment information is shown in table 1.
TABLE 1 high response peptide fragment information
4. Peptide fragment selection validation
On the basis of qualitative analysis, peptide segments with good reproducibility and high response intensity are selected and targeted verification is carried out by adopting a Nano LC-QE-Orbitrap-MS PRM mode.
The samples are injected into a Nano LC-QE-Orbitrap-MS system, and high-resolution mass spectrum data of the samples are collected. Water (containing 0.1% formic acid) is used as a flow line A, acetonitrile (containing 0.1% formic acid) is used as a mobile phase B, and the mobile phase A and the mobile phase B are mixed to obtain an AB mixed solution for gradient elution, wherein the elution procedure is as follows: 0-2min,2% -5%; 2-48min,5% -32% B; 48-53min,32% -45% by weight B;53-55min,45% -100% B;55-60min,100% B. The acquisition time of the atlas is 60min. The mass spectrometry detection adopts a PRM scanning mode, a primary mass spectrometry is a positive ion mode, the scanning range m/z is 150-2,000, the resolution is 70,000, the automatic gain control target (AGC target) is 3 × e6, and the Maximum residence time (Maximum IT) is 200ms. PRM scan resolution 17500, automatic gain control target (AGC target) 2 × e5, maximum dwell time (Maximum IT) 100ms, isolation width of parent ion (Isolation window) 2m/z, normalized collision energy setting 27.
5. Peptide fragment selection
According to the PRM validation results, AVFPSIVGRPR (shown as PEP 1), PEPAAKSAPAPK (shown as PEP 2), GIADALGKAYH (shown as PEP 3), GVTIAQGGVLPNIQ (shown as PEP 4) were selected as target analytes. Peptide fragments GIADALGKAYH (shown as PEP 3), GVTIAQGGVLPNIQ (shown as PEP 4) are double-charge ions, AVFPSIVGRPR (shown as PEP 1) and PEPAAKSAPAPK (shown as PEP 2) are triple-charge ions, and the molecular weight is in the range of 1091.60-1365.76 Da. The information of 4 target peptide fragments of PEP 1-PEP 4 is described in Table 1, and the mass spectrum is shown in FIG. 2.
2. Quantitative analysis of polypeptide component of cinobufagin preparation
1. Sample solution preparation
1.1 preparation of Standard solutions
Respectively preparing stock solutions of AVFPSIVGRPR (shown as PEP 1), PEPAAKSAPAPK (shown as PEP 2), GIADALGKAYH (shown as PEP 3) and GVTIAGGVLPNIQ (shown as PEP 4) with pure water to concentration of 5mg/ml, and storing at-80 deg.C.
Precisely sucking stock solutions GVTIAQGGVLPNIQ (shown as PEP 4) of each standard product to prepare 4 peptide fragment mixed standard solution with the concentration of 100 mu g/mL, AVFPSIVGRPR (shown as PEP 1), PEPAAKSAPAPK (shown as PEP 2) and GIADALGKAYH (shown as PEP 3) to prepare 4 mu g/mL, and gradually diluting the mixed standard solution by 5 times by using water (containing 0.1% formic acid) to obtain standard solutions with series concentrations.
1.2 preparation of test solutions
1.2.1 preparation of Cinobufagin preparation samples
Taking a cinobufagin preparation sample, concentrating and volatilizing the cinobufagin preparation sample, adding 0.1% formic acid-water for redissolving to prepare a 10mg/mL solution to be detected of the cinobufagin preparation. Adding a certain volume of cinobufagin preparation solution to be tested (the amount of polypeptide is 20-200 mug) into a certain volume of dithiothreitol (the final concentration is 5 mM) respectively, incubating for 1h at 37 ℃ in a constant-temperature mixing instrument, adding a certain volume of iodoacetamide (the final concentration is 0.15M), and incubating for 45min at 37 ℃ in a dark place. Desalting the sample with desalting column, mixing, volatilizing, and re-dissolving with 0.1% formic acid-water to obtain cinobufagin preparation sample solution.
1.2.2 toad skin sample preparation
Weighing toad skin, cutting into pieces, adding 10mL of ultrapure water, decocting for 45min, sieving with a No. 6 sieve, adding 10mL of ultrapure water into the residue, decocting for 30min, sieving with a No. six sieve, mixing the filtrates, and concentrating. Adding ethanol into the concentrated solution to adjust the ethanol content to 60%, mixing, standing at 4 deg.C for 24 hr, filtering to obtain supernatant, and recovering ethanol until no ethanol smell exists. Adding ethanol again to adjust alcohol content to 85%, mixing, standing at 4 deg.C for 48 hr, filtering to obtain supernatant, and recovering ethanol until no alcohol smell is obtained, i.e. cutis Bufonis polypeptide extract.
Adding a certain volume of toad skin polypeptide extract (with a polypeptide content of 20-200 μ g) into a certain volume of dithiothreitol (with a final concentration of 5 mM) respectively, incubating at 37 ℃ for 1h in a constant-temperature mixer, adding a certain volume of iodoacetamide (with a final concentration of 0.15M) into the constant-temperature mixer, and incubating at 37 ℃ in a dark place for 45min. Desalting the sample with desalting column, mixing, volatilizing, and re-dissolving with 0.1% formic acid-water to obtain toad skin sample solution.
2. Establishing a standard curve equation: injecting the series of standard substance solutions obtained in the step 1.1 into a liquid chromatography-mass spectrometer for analysis, and drawing a standard curve equation with the peak area as a vertical coordinate and the concentration as a horizontal coordinate as shown in table 2;
TABLE 2 Linear equation, linear Range, correlation coefficient for 4 peptides
| PEP No. | Peptide fragment sequence | Regression equation | R 2 | |
| PEP | ||||
| 1 | AVFPSIVGRPR | y＝217.318x+5513.62 | 0.9985 | 6.4ng/mL～4μg/ |
| PEP | ||||
| 2 | PEPAKSAPAPK | y＝326.786x-35487.9 | 0.9968 | 12.8ng/mL～4μg/ |
| PEP | ||||
| 3 | GIADALGKAYH | y＝1182.24x-56961.1 | 0.9990 | 6.4ng/mL～4μg/mL |
| PEP 4 | GVTIAQGGVLPNIQ | y＝3547.23x-10284.5 | 0.9901 | 0.32μg/mL～100μg/mL |
3. Content determination: and (3) injecting the cinobufagin preparation sample solution or the toad skin sample solution obtained in the step (1.2) into a liquid chromatography-mass spectrometer, and calculating the content of the peptide fragments shown by PEP 1-PEP 4 according to the standard curve equation obtained in the step (3).
The cinobufotalin preparation and the toad skin are subjected to synchronous quantitative analysis of 4 peptide fragments, and the quantitative results are shown in table 3. The result shows that the content of the target peptide fragments of the preparation batches is different, the content basically shows that the content is PEP 4 > PEP 1 > PEP 2 > PEP 3, the content of the peptide fragment 4 is the highest, and the content of the peptide fragment 3 is the lowest. The same trend is shown in toad skin. In addition, the content of the target peptide fragment in the toad skin is far higher than that in the preparation. The detection of the corresponding peptide fragment is prompted, and the component analysis, identification and quality control of the cinobufagin preparation can be realized.
TABLE 3 Cinobufotalin preparations and the content of target peptide fragments in cutis Bufonis (ng/mL, μ g/g)
| Sample(s) | Lot/place of | PEP | 1 | |
|
PEP 4 |
| |
210704 | 39.59ng/mL | 20.69ng/mL | 14.45ng/mL | 536.08ng/ | |
| Cinobufotalin preparation | ||||||
| 2 | 210708 | 42.87ng/mL | 19.80ng/mL | 11.31ng/mL | 616.79ng/mL | |
| Toad skin 1 | (Anhui) | 0.40μg/g | 0.36μg/g | 0.57μg/g | 22.71μg/ | |
| Toad skin | ||||||
| 2 | Guangdong (Chinese character of 'Guangdong') | 0.30μg/g | 0.42μg/g | 0.68μg/g | 18.60μg/g |
The chromatographic conditions of the above steps are as follows:
a synergy TM Fusion-RP chromatography column (50 x 2mm,2.5 μm,
). The mobile phase A is an aqueous solution containing 0.1% formic acid, and the phase B is acetonitrile containing 0.1% formic acid. Liquid phase gradient: 0-1min,60% -90%, B,1-1.5min,90% -90%, B,1.5-2min,90% -60%. Sample injection amount: 2 μ L. Flow rate: 0.3mL/min. Column temperature: at 40 ℃.
The mass spectrum conditions are as follows:
the mass spectrometry conditions were multiple reaction monitoring mode (MRM), with detection in positive ion mode. The specific instrument parameters are as follows: the spraying voltage is 3500V, the evaporation temperature is 300 ℃, the sheath gas is 45psi, the ion scanning air pressure is 2.0psi, the auxiliary gas is 15psi, and the capillary temperature is 270 ℃.
4. Online optimization of mass spectrometry parameters
The method establishment process is briefly described by taking the peptide segment AVFPSIVGRPR (as shown in PEP 1) as an example. With its triply charged excimer ion ([ M + 3H)] 3+ ) m/z 400.67 is parent ion, single charge fragment ions m/z 70.1, 120.1 and 72.2 construct three pairs of ion pairs, m/z 400.67>70.1，400.67>120.1，400.67>72.2, different ions generate different collision energies, a Gaussian curve fitting is utilized to construct a candidate ion pair relative response value-collision energy curve, and the CE value corresponding to the highest point of the curve is used as the optimal CE value; different parent ions corresponding to different ion introduction ring projection frequency amplitudes (S-Lens RF) are utilized to construct candidates by Gaussian curve fittingAnd taking the S-Lens RF value corresponding to the highest point of the curve as the optimal S-Lens RF value. The quantitative ion pairs and mass spectral parameters of the 4 target peptide fragments can be seen in table 4.
TABLE 4 sequences of target peptides and conditions for mass spectrometric detection
5. Methodology validation
The linear result shows that the correlation coefficients of the 4 peptide segments are all larger than 0.99, which indicates that the peptide components have good linear relation, and in methodology investigation, the repeatability experiment RSD is less than 3 percent, which indicates that the repeatability of the method is good; and has good stability experiment and precision.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is intended to be covered by the present invention.
Sequence listing
<110> Nanjing university of traditional Chinese medicine
<120> a characteristic polypeptide for identifying cinobufagin preparation or toad skin, and content determination method and application thereof
<141> 2022-05-24
<160> 25
<170> SIPOSequenceListing 1.0
<210> 2
<211> 11
<212> PRT
<213> Chinese toad (Bufo Bufo gargargargarizans)
<400> 2
Ala Val Phe Pro Ser Ile Val Gly Arg Pro Arg
1 5 10
<210> 2
<211> 11
<212> PRT
<213> Chinese toad (Bufo Bufo gargargargarizans)
<400> 2
Pro Glu Pro Ala Lys Ser Ala Pro Ala Pro Lys
1 5 10
<210> 2
<211> 11
<212> PRT
<213> Chinese toad (Bufo Bufo gargargargarizans)
<400> 2
Gly Ile Ala Asp Ala Leu Gly Lys Ala Tyr His
1 5 10
<210> 2
<211> 14
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln
1 5 10
<210> 2
<211> 12
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Ala Pro Gly Gln Pro Gln Leu Gln Ile Ser Gly Gln
1 5 10
<210> 2
<211> 10
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Ala Pro Gly Pro Lys Gly Glu Arg Gly Leu
1 5 10
<210> 2
<211> 10
<212> PRT
<213> Chinese toad (Bufo Bufo gargargargarizans)
<400> 2
Ala Ser Thr Lys Glu Lys Leu Ile His Asn
1 5 10
<210> 2
<211> 12
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Pro Pro Pro Gly Pro Gly Gly Pro Tyr Pro Gly Pro
1 5 10
<210> 2
<211> 25
<212> PRT
<213> Chinese toad (Bufo Bufo gargargargarizans)
<400> 2
Ala Gly Glu Lys Val Ile Lys Glu Thr Gly Gly Ile Ile His Gly Ile
1 5 10 15
Gly Asp Ala Ile Gly Gly Leu Gly Lys
20 25
<210> 2
<211> 13
<212> PRT
<213> Chinese toad (Bufo Bufo gargargargarizans)
<400> 2
Pro Ile Phe Pro Ala Ala Pro Ala Ser Met Gly Phe Asn
1 5 10
<210> 2
<211> 15
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Glu Glu Glu Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met
1 5 10 15
<210> 2
<211> 13
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Pro Val Pro Tyr Trp Glu Val Gln Pro Ala Thr Phe Arg
1 5 10
<210> 2
<211> 12
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Ala Ser Leu Gln Leu Ile Asn Val Asn Phe Gln Gln
1 5 10
<210> 2
<211> 10
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Pro Thr Thr Glu Phe Leu Leu Lys Ala Asn
1 5 10
<210> 2
<211> 21
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Pro Thr Glu Phe Pro Ala Leu Thr Ala Glu Gln Lys Lys Glu Leu His
1 5 10 15
Asp Ile Ala Gln Arg
20
<210> 2
<211> 21
<212> PRT
<213> Chinese toad (Bufo Bufo gargargargarizans)
<400> 2
Ser Arg Gln Ser Thr Tyr Thr Lys Thr Phe Ser Ala Ala Ser Ala Val
1 5 10 15
Gln Pro Arg Pro Asn
20
<210> 2
<211> 11
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe
1 5 10
<210> 2
<211> 12
<212> PRT
<213> Chinese toad (Bufo Bufo gargargargarizans)
<400> 2
Pro Val Ile Leu Gly Lys Tyr Pro Asn Ser Phe Thr
1 5 10
<210> 2
<211> 21
<212> PRT
<213> Chinese toad (Bufo Bufo gargargargarizans)
<400> 2
Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe Pro Ser Ile
1 5 10 15
Val Gly Arg Pro Arg
20
<210> 2
<211> 13
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Ala Thr Gly Gln Ala Gly Pro Pro Gly Pro Val Gly Pro
1 5 10
<210> 2
<211> 13
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Pro Val Gln Tyr Trp Glu Ile Gln Pro Ala Thr Phe Arg
1 5 10
<210> 2
<211> 17
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
His Ile Gln Glu Val Ala Ala Glu Ser Val Gly Thr Gly Ile Met Gly
1 5 10 15
Lys
<210> 2
<211> 21
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Lys Pro Gln Asp Asp Ser Ala Asn Ala Gly Gly Ile Ser Ser Thr Ser
1 5 10 15
Ala Ser Val Asn Arg
20
<210> 2
<211> 20
<212> PRT
<213> Chinese toad (Bufo Bufo gargargargarizans)
<400> 2
Thr Ser Gln Tyr Ala Arg Ser Leu Gly Gly Gly Gln Ser Ala Gly Ser
1 5 10 15
Ser Ser Phe Arg
20
<210> 2
<211> 22
<212> PRT
<213> China big toad (Bufo Bufo gargargarizans)
<400> 2
Leu Asp Arg Leu Glu Gly Gly Ser Leu Ser Gln Ala Ser Ser Met Val
1 5 10 15
Ala Ser Gly Ala Asp Gly
20
Claims (10)
1. A polypeptide for identifying cinobufotalin preparation or toad skin, characterized by: the characteristic polypeptide comprises one or more of PEP 1-PEP 25; the sequence of PEP 1-PEP 25 is AVFPSIVGRPR, PEAKSAPAPK, GIADALGKAYH, GVTIAGGVLPNIQ, AP (+ 15.99) GQPQLQISGQ, APGP (+ 15.99) KGERGL, A (+ 42.01) STKEKLIHN, PPPGPGGPYPGP, AGEKVIKETGGIGIGDAIGGLGLK, PIFPAAPASM (+ 15.99) GFN, E (+ 42.01) EEIAVVSDNGSGM (+ 15.99), PVPYWEVQPATFR, A (+ 42.01) SLQQVNQQQQQQQQQQQQQQFNAN, PTFPALQAEKKELHDAQR, S (+ 42.01) RQSSTKTAAVQPN, WGIQFLQFN, WGIQFLQFNGT, PVGPNTGPT, PTEGPRGGPRGGPRG, PTQAGFPAGASTGASTGASTGS (+ 15.15) GGAGASTGASTGASTGS (+ 15.15.15.
2. The polypeptide of claim 1, wherein the polypeptide is AVFPSIVGRPR (PEP 1), PEPAAKSAPAPK (PEP 2), GIADALGKAYH (PEP 3), GVTIAQGGVLPNIQ (PEP 4).
3. A quantitative analysis method for identifying a characteristic polypeptide of a cinobufagin preparation or toad skin, characterized by: the method comprises the following steps:
(1) Preparation of a standard solution: preparing a mixed standard solution by taking characteristic polypeptides PEP 1-PEP 4, namely AVFPSIVGRPR, PEPAAKSAPAPK, GIADALGKAYH and GVTIAQGGVLPNIQ, and diluting to obtain a series of standard solutions with the concentration range of 6.4 ng/mL-100 mu g/mL;
(2) Preparation of a test solution: extracting cinobufagin preparation polypeptide or toad skin test solution;
(3) Establishing a standard curve equation: injecting the series of standard substance solutions in the step (1) into a liquid chromatography-mass spectrometer for analysis, and drawing a standard curve equation by taking the peak area as a vertical coordinate and the concentration as a horizontal coordinate;
(4) Content determination: and (3) injecting the cinobufagin preparation polypeptide or toad skin test solution obtained in the step (2) into a liquid chromatography-mass spectrometer, and calculating the content of the peptide segments shown by PEP 1-PEP 4 according to the standard curve equation obtained in the step (3).
4. The quantitative analysis method for identifying a polypeptide characteristic of a cinobufagin preparation or toad skin according to claim 3, wherein: step (1) preparation of standard solution: respectively taking AVFPSIVGRPR, PEPAAKSAPAPK, GIADALGKAYH and GVTIAQGGVLPNIQ, respectively preparing standard stock solutions with concentration of 5mg/ml with pure water, and storing at-80 deg.C;
then precisely absorbing each standard substance stock solution to prepare 4 characteristic peptide fragment mixed standard substance solutions with the GVTIAQGGVLPNIQ concentration of 100 mu g/mL and the AVFPSIVGRPR, PEPAAKSAPAPK and GIADALGKAYH concentration of 4 mu g/mL, and gradually diluting the mixed standard substance solutions by 5 times by using 0.1% formic acid water to obtain standard solutions with serial concentrations.
5. The quantitative analysis method for identifying a polypeptide characteristic of a cinobufagin preparation or toad skin according to claim 3, wherein: the preparation method of the test solution in the step (2) comprises the following steps:
taking a cinobufagin preparation sample, taking supernatant for ultrafiltration treatment, concentrating and drying ultrafiltration liquid, and preparing a cinobufagin preparation extracting solution of 5 mg/mL-15 mg/mL; sequentially carrying out disulfide reduction and alkylation treatment on the extracting solution, volatilizing, and re-dissolving with formic acid-water to obtain a cinobufagin preparation test sample solution;
or weighing toad skin, shearing, adding ultrapure water, decocting, mixing decoction, extracting filtrate, concentrating, adding ethanol into the concentrated solution to adjust the ethanol content to 60-70%, mixing uniformly, standing, filtering supernatant, and recovering ethanol until no ethanol smell exists; adding ethanol again to adjust the alcohol content to 80-85%, mixing uniformly, standing, filtering out supernatant, and recovering ethanol until no alcohol smell exists, namely the toad skin polypeptide extract; adding a certain volume of toad skin polypeptide extract into a disulfide reducing agent respectively, incubating in a constant-temperature mixing instrument, adding an alkylating reagent, incubating in a constant-temperature mixing instrument in a dark place, desalting a sample by using a desalting column, combining and volatilizing, and finally re-dissolving with formic acid-water to obtain a toad skin test solution.
6. The quantitative analysis method for identifying a polypeptide characteristic of a cinobufagin preparation or toad skin according to claim 4, wherein: the disulfide reducing agent comprises dithiothreitol; the alkylating agent includes iodoacetamide.
7. The quantitative analysis method for identifying a polypeptide characteristic of a cinobufagin preparation or toad skin according to claim 3, wherein: the chromatographic conditions of the liquid chromatogram-mass spectrometer analysis in the step (3) and the step (4) are as follows: adopting a polar group bonded C18 chromatographic column, carrying out gradient elution by using 0.05-0.2% formic acid-water solution as a mobile phase A and 0.05-0.2% formic acid-acetonitrile solution as a mobile phase B at the flow rate of 0.1-0.5 mL/min, wherein the elution procedure is as follows: 0-1min, 60-90% by weight B,1-1.5min, 90-90% by weight B,1.5-2min, 90-60% by weight B; sample introduction amount: 2 mu L of the solution; flow rate: 0.3mL/min, column temperature: 30-50 ℃;
the mass spectrum conditions are as follows: adopting ESI positive ionization mode and SRM scanning mode; the source parameters of mass spectrum detection ESI positive ionization mode are as follows: the spraying voltage is 3500V, the evaporation temperature is 300 ℃, the sheath gas pressure is 45psi, the ion scanning air pressure is 2.0psi, the auxiliary gas pressure is 15psi, and the capillary temperature is 270 ℃.
8. The quantitative analysis method for identifying a polypeptide characteristic of a cinobufagin preparation or toad skin according to claim 3, wherein: the standard curve equation and the linear range established in the step (3) are as follows:
。
9. A kit for identifying cinobufotalin preparation or toad skin is characterized by comprising the following components in parts by weight: it comprises the signature polypeptide of claim 1 or 2.
10. Use of the signature polypeptide of claim 1 or 2 in cinobufagin analysis, cinobufagin identification and cinobufagin quality control.
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