Marker polypeptide of bothrops atrox-like thrombin and method thereof for detecting species source and content of snake venom-like thrombin and application

A method for detecting a species source of a snake venom-like thrombin, wherein the following steps are used:

• (1) adding a trypsin to a sample to be detected to perform enzymolysis pre-treatment, and taking a supernatant;
• a specific operation is as follows:
• taking 20 mg of the sample to be detected, and then adding an ammonium bicarbonate solution with a concentration of 25 mmol/L and fixing the volume to 100 mL, and shaking uniformly, to obtain a solution;
• measuring 1 mL of the solution, adding 10 μL of a trypsin solution with a concentration of 10 mg/mL, reacting at 37° C. for 4 hours, and taking the supernatant by centrifugation;
• (2) injecting the supernatant into a liquid chromatography-mass spectrometer respectively, using an electrospray positive ion mode to perform a multi-reaction monitoring, using a mass-to-charge ratio double-charge of 481.9 to 485.2 as a qualitative ion pair, and using a mass-to-charge ratio double-charge of 481.9 to 315.2 as a quantitative ion pair, to detect the supernatant;
• in detection conditions of a liquid phase and a mass spectrometry in the liquid chromatography-mass spectrometer, the liquid phase conditions are:
• Thermo Hypersil GOLD C18 chromatographic column (100 mm×2.1 mm, 3 μm);
• column temperature:
• 40° C.;
• injection volume:
• 10 μL;
• flow rate:
• 0.3 mL/min;
• a mobile phase A is 0.1% of a formic acid solution, B is 0.1% of a formic acid acetonitrile, and gradient elution is performed, elution program:
• 0 to 2 min, mobile phase A 90%;
• 2 to 6 min, mobile phase A 90% to 60%;
• 6.1 to 8 min, mobile phase A 30%;
• and 8.1 to 10 min, mobile phase A 90%;
• in the detection conditions of the liquid phase and the mass spectrometry in the liquid chromatography-mass spectrometer, the mass spectrometry conditions are:
• an electrospray ion source, a positive ion scanning mode, and the multi-reaction monitoring;
• a vortex ion spray temperature is 500° C.;
• an ionization voltage is 5.5 kV;
• a collision chamber exit potential is 10 V;
• an entry potential is 10 V;
• a collision energy is 20 V, and a declustering potential is 80 V;
• (3) if a chromatogram obtained in the step (2) shows a chromatographic peak consistent with a retention time of a control marker polypeptide chromatogram, it is indicated that the sample to be detected is from Bothrops atrox;
• and otherwise, it is not a source of the Bothrops atrox, wherein an amino acid sequence of the control marker polypeptide is EAYNGLPAK (SEQ ID NO:
• 1).