Quantitative detection method for snake venom thrombin-like enzyme (svtle)

A quantitative detection method for snake venom thrombin-like enzyme (SVTLE), comprising the following specific steps of:

• (1) taking a reference substance of a marker peptide for SVTLE from Agkistrodon halys pallas with an amino acid sequence of LDSPVSNSAHIAPLSLPSSAPSVGSVCR, and dissolving and diluting the reference substance to prepare a series of reference solutions with different concentrations;
• (2) taking a to-be-detected sample, and dissolving the to-be-detected sample to prepare a test solution;
• (3) taking the series of reference solutions with different concentrations in step (1), adding the test solutions in step (2) respectively for uniform mixing, conducting enzymolysis with trypsin, and then taking supernatants after enzymolysis as a series of solutions to be detected;
• (4) injecting the solutions to be detected in step (3) into a liquid chromatogram-mass spectrometer respectively, conducting multiple-reaction monitoring by adopting an electrospray positive ion mode, and conducting detection with tricharged 935.8→602.3 as a qualitative ion pair and tricharged 935.8→861.4 as a quantitative ion pair;
• and
• (5) extracting an ion pair tricharged 935.8→861.4 chromatogram, calculating a linear regression equation y=ax+b (r>0.99) with an addition quantity of the marker peptide in the series of solutions to be detected as a horizontal coordinate and a corresponding peak area as a vertical coordinate, and then calculating an x value when y=0 according to the regression equation to obtain the quantity of the marker peptide in the solutions to be detected, and then calculating contents of the marker peptide of the SVTLE from Agkistrodon halys pallas in the solutions to be detected;
• wherein liquid phase and mass spectrum detection conditions in the liquid chromatogram-mass spectrometer are as follows:
• the Liquid phase conditions:
• Waters ACQUITY UPLC BEH C18 chromatographic column:
• 50 mm×2.1 mm, 1.7 μm particle size;
• column temperature:
• 40° C.;
• sample size:
• 2 μL;
• flow rate:
• 0.2 mL/min;
• a mobile phase A is 0.1% formic acid solution and a mobile phase B is 0.1% formic acid acetonitrile for gradient elution, a elution procedure of which is as follows:
• 0→1 min, the mobile phase A 80%;
• 1→5 min, the mobile phase A 80%→10%;
• 5→7 min, the mobile phase A 10%→10%;
• and
• the Mass spectrum conditions:
• ESI source, positive ion scanning mode, multiple-reaction monitoring;
• eddy ion spraying temperature:
• 500° C.;
• deionization voltage:
• 5.5 kV;
• exit potential of a collision chamber:
• 10 V;
• entrance potential (EP):
• 10 V, declustering potential (DP):
• 135 V;
• and by mass-to-charge ratio (m/z), tricharged 935.8→602.3 is taken as the qualitative ion pair with collision energy of 40 V, and tricharged 935.8→861.4 is taken as the quantitative ion pair with collision energy of 45 V.