[Determination of the species origin and thrombin-like enzyme content of Bothrops atrox venom by ultra-high performance liquid chromatography-tandem mass spectrometry based on marker peptide]

Abstract

Snake venom thrombin drugs are hemostatic drugs prepared from Agkistrodon halys venom, and the main active ingredients are snake venom thrombin-like enzymes (svTLEs). The svTLEs derived from different snake species differ in their structures, hemostatic mechanisms, and pharmacological effects. Therefore, accurate identification of the source of snake venom species and determination of the svTLE content are essential to ensure the quality of these products. Based on proteomics technology, the marker peptides of svTLEs from Bothrops atrox were screened with species specificity for the first time in this study, and an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for species identification and determination of the svTLE content of Bothrops atrox was established. After reductive alkylation and trypsin enzymolysis of the purified svTLE from Bothrops atrox, enzymatic peptide fragments were obtained and determined by easy-nano liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (Nano LC-Q-Exactive-MS). The mass spectrum data were analyzed by Proteome Discoverer 2.2 software. The maker peptide "EAYNGLPAK", which characterized the svTLE from Bothrops atrox, was finally screened and validated by comparison of the basic local alignment search tool (BLAST) with the NCBI and UniProt databases. For the marker peptide, the enzymolysis temperature, enzymolysis time and amount of enzyme for the sample preparation were optimized. The optimized enzymolysis conditions were as follows: enzymolysis temperature, 37 ℃; enzymolysis time, 4 h; and amount of enzyme, 10 μL. A qualitative and quantitative detection method based on UHPLC-MS/MS was established by optimizing the chromatographic and mass spectrometric conditions. Accordingly, 20 mg of the evenly mixed sample was weighed and placed in a 100 mL volumetric flask. Then, 25 mmol/L ammonium bicarbonate solution was added to dissolve the sample, and the solution was diluted to the scale. Precisely 1.00 mL of the solution was extracted; subsequently, addition of 10 μL trypsin solution was added, followed by shaking, and the mixture was placed in an incubator for 4 h to induce enzymolysis at a constant temperature of 37 ℃. The mixture was subsequently removed from the incubator, cooled to ambient temperature, centrifuged at 12000 r/min for 10 min, and analyzed by LC-MS. Separation was performed on the UPLC system with a Thermo Hypersil GOLD C18 column (100 mm×2.1 mm, 3.0 μm) under the gradient elution of acetonitrile containing 0.1% (v/v) acetic acid and water containing 0.1%(v/v) acetic acid, at a flow rate of 0.3 mL/min, column temperature of 30 ℃, and injection volume of 2 μL. The maker peptides were determined in the electrospray positive ionization (ESI⁺) and multiple reaction monitoring (MRM) modes using the external standard curve method. The detection ions were m/z 481.9> 315.2 and 481.9> 485.2. There was a good linear relationship between the mass concentration of the marker peptide and the chromatographic peak area in the range of 2.5-30 ng/mL, and the correlation coefficient (r) was 0.9996, The limit of detection (S/N=3) and limit of quantification (S/N=10) were 2.5 mg/kg and 6.25 mg/kg, respectively. At spiked levels of 40, 80, and 120 mg/kg, the recoveries of the marker peptides were 95.5%-101.9%, while the relative standard deviations (RSDs) of the results for parallel analyses at various spiked levels were 1.1%-3.2%. The developed method is simple, rapid, sensitive, and specific, and it can be used for the identification of Bothrops atrox venom species and determination of the svTLE content. The findings of this study would help ensure the quality of hemocoagulase products from the relevant source and provide a reference for the quality control of other snake venom products.

Keywords: Bothrops atrox; marker peptide; snake venom; snake venom thrombin-like enzymes (svTLEs); ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).

图 1. 特征肽(EAYNGLPAK)的一级和二级质谱图

图 2. 不同酶解时间下监测离子的峰面积

图 3. 不同酶用量下监测离子的峰面积

图 4. 特征肽对照品溶液(10 ng/mL)、空白溶液及阴性样品溶液的MRM色谱图