A TYPE OF HEMOCOAGULASE MARKER PEPTIDE OF AGKISTRODON HALYS AND THE APPLICATION IN IDENTIFYING THE SPECIES OF HEMOCOAGULASE FOR INJECTION

• Main Authors: Hang, Baojian, Shi, Feng, Gong, Liping, Wang, Weijian, You, Pengfei, Hu, Defu, Xian, Ruiqing, Wang, Congcong
• Format: Patent
• Language: English
• Published: 22.04.2021
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; COMPOSITIONS THEREOF; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; CULTURE MEDIA; ENZYMOLOGY; INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIRCHEMICAL OR PHYSICAL PROPERTIES; MEASURING; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; ORGANIC CHEMISTRY; PEPTIDES; PHYSICS; PROCESSES OF PREPARING SUCH COMPOSITIONS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; TESTING; VINEGAR; WINE

This invention provides a type of hemocoagulase marker peptide of Agkistrodon halys and the application in identifying the species of hemocoagulase for injection, in which the sample to be tested and the reference substance of the marker peptide are respectively dissolved into the test solution and the reference solution, and then processed with dithiothreitol and iodoacetamide for alkylation reduction. After adding ammonium bicarbonate solution for dilution, add enzyme for hydrolysis, and then centrifuge at a high speed, to get the supernatant, and then add it to the liquid chromatography-mass spectrometer for analysis. Based on enzymolysis, liquid chromatography-tandem mass spectrometry-multi-reaction monitoring (LC-MS/MS-MRM) method, extract the ion flow chromatography by using the three-charge 935.8-861.4 and three-charge 935.8-602.3 as the detection ion pairs, and identify the species of the hemocoagulase marker peptide of Agkistrodon halys for injection. This method is simple, convenient, and highly exclusive, filling in the blanks in identifying the source of species of the hemocoagulase marker peptide of Agkistrodon halys, and improving the quality control level as well. i XCof+MRM(4pai):93.300/602.00 DaomSample119(ZJ 0.02/0.0) of20200520.ff (TrboSpraylo rve)M . 1.1- tt4Wes-m/z:935.8/602.3 O000A Reference substance of marker peptide -4000-00] 0.0 U. L.. l,5 210 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Tim mi XSCof+MRM(4pa ):93.00 /861.00 ao Sam ple 119(ZJ 0.02/0.06)of20200520.wif(TurboSpraymlo e) Mao.9992.00cp MOO] WIEtiEf et4 71-m/z:935.8/861.4 600o Reference substance of marker peptide o4000 .0 0.5 LA L.5 20 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Figure 4 SXIC ofMRM (4 pai): 936.300 02500 Da rSample 128 (ZJ 2) of 20200520.iff (Tr Spray lo0rive) .1.8ep s- a ffl n M CAT h -m/z:935.8/602.3 - Agkistrodon halys hemocoagulase for injection 5000.00 . O 0 40 O 00 i 0.0 0.5 L 1 1 2.0 2.5 3.0 13 5 40 4.5 5.0 5.5 Time, m XCof+MRM(4pa ):936.3000.00ao rSample128(ZJ2)of20200520wiff(Tro SprayloDrie) 1. p 5.Itffl rHIM Uf&N7 -m/z:935.8/861.4 Agkistrodon halys hemocoagulase for injection 50000o .. 2. 2.5 3.0 35 4,0 4.5 5. Time-in Figure 5 M xAC of +MRM (4 par,):936.3001602500 D, f-o Sample 247 (Mao_1) of 20200520..wi«(T.rb SpranosiDve)Mx.600e. 3-~~RM W tf, H l Wl R-m/z:935.8/602.3 3- Bothrops jararaca hemocoagulase for injection 0. .5 1L 2A 2.5 3.0 3.5 40 4. 50 . Time mi M XAC of +MRM (4 pairs): 936.300/81.50 Da fro Sample 247 (Mao_1) of202000wi (Turbo Spray 1,nDrive) Ma. 6.2e4 p. 6mfl fMW I.KiLN?-m/z:935.8/861.4 Bothrops jararaca hemocoagulase for injection 0.0 05 1 0 L5 20 2,S 3I0 3I5 4,0 415 5 11S Time-in Figure 6