Quantitative detection method of venom thrombin-like enzyme
A quantitative detection method and thrombin-like technology, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, measuring devices, etc., can solve the problems of cumbersome determination methods, poor accuracy, and the inability to accurately characterize the quality of snake venom, and achieve improvement. The level of quality control, quantitative accuracy, and the effect of filling the gaps in quality standards
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[0031] The preparation method of relevant reagent reagent and solution in the following examples is as follows:
[0032] (1) Reagents: trypsin (Sigma, batch number SLBS8956), Agkistrodon haemagglutinate (Jinzhou Aohong Pharmaceutical Co., Ltd., purity 98.5%), guanidine hydrochloride (VETEC, batch number WXBC4261V), trihydroxyaminomethane (Shanghai Stock Exchange , batch number 20181206), dithiothreitol (BBI Life Sciences, batch number D911BA0011), iodoacetamide (BBI Life Sciences, batch number B326BA1943), and other reagents were of analytical grade.
[0033] (2) 25 mmol / L ammonium bicarbonate solution: Weigh 79.06 mg of ammonium bicarbonate, add 40 mL of water to dissolve, and obtain.
[0034] (3) Dithiothreitol (DTT) solution: Weigh 15.42 mg of dithiothreitol and add 500 μL of water to dissolve it.
[0035] (4) Iodoacetamide (IA) solution (newly prepared before use): Weigh 18.5 mg of iodoacetamide, add 500 μL of water to dissolve, and obtain the solution.
[0036] (5) 0.4 ...
Embodiment 1
[0038] Screening and Determination of Characteristic Peptides of Thrombin in Agkistrodon halys
[0039] 1 Instruments and equipment
[0040] Thermo Fusion high-resolution mass spectrometer (Thermo Fisher Scientific, USA), EASY-nLC 1000 nanoliter liquid chromatograph (Thermo Fisher Scientific, USA), CP225D electronic balance (Sartorius, Germany), Sigma 3-30 K refrigerated centrifuge ( Sigma, Germany), Milli-QAdvantage A10 ultrapure water meter (Millipore, USA).
[0041] 2 Chromatography and mass spectrometry conditions
[0042] Chromatographic column: 0.2 mm × 3.5 cm (5 μm particle size) ReproSil-Pur C made in the laboratory 18-AQ Trapcolumn desalted and enriched, using a laboratory-made 75 μm × 25 cm (3 μm particle size) ReproSil-Pur C18-AQ nanoliter analysis column for separation. Mobile phase A was 2% acetonitrile in 0.1% formic acid in water; mobile phase B was 98% acetonitrile in 0.1% formic acid in water. The flow rate of the nanoliter separation pump is 300 nL / min, a...
Embodiment 2
[0050] Multiple reaction monitoring (MRM) was used to quantify the thrombin in the samples of Agkistrodon halys.
[0051] 1. Instruments and equipment
[0052] SCIEX Triple Quad 6500 triple quadrupole mass spectrometer, CP225D electronic balance (Sartorius, Germany), Sigma 3-30 K refrigerated centrifuge (Sigma, Germany), Milli-QAdvantage A10 ultrapure water instrument (Millipore, USA).
[0053] 2. Chromatography and mass spectrometry conditions
[0054] Liquid phase conditions: Waters ACQUITY UPLC BEH C 18 Chromatographic column (50 mm×2.1 mm, 1.7 μm); column temperature: 40°C; injection volume: 2 μL; flow rate: 0.2 mL / min; mobile phase A is 0.1% formic acid, B is 0.1% formic acid in acetonitrile, Gradient elution, elution program: 0→1 min, mobile phase A 80%; 1→5 min, mobile phase A 80%→10%; 5→7 min, mobile phase A 10%→10%.
[0055] Mass spectrometry conditions: electrospray ion source, positive ion scanning mode, multiple reaction monitoring; vortex ion spray temperature...
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