| Snake venom is a liquid rich in proteins and peptides secreted from venom glands,which account for about 90～95%of the dry weight of venom.Modern pharmacology shows that snake venom plays a key role in the physiological processes of cell apoptosis,neurotransmission,hemostasis,and signal transduction,and has the characteristics of high biological activity and high target specificity.Due to its advantage of high druggability,the probability of developing new drugs from snake venom is more than1000 times that of synthetic compounds,has become one of the research hotspots of new drug development in recent years.However,result of the snake venom proteins and peptides variety,and the the content is very small,the research of snake venom proteomics faces many technical challenges.At present,the separation mode based on a single mechanism cannot meet the complete separation of all components in snake venom.In recent years,two-dimensional gel electrophoresis technology(2-DE)based on separation mechanism of protein isoelectric point and molecular size has been widely used in the separation and analysis of snake venom toxins,but there are a series of shortcomings such as the inability to detect proteins with small molecular weight and extreme isoelectric point,poor reproducibility,time-consuming,labor-intensive operation,and cumbersome operation.Two-dimensional liquid chromatography(2D-LC),by combining the chromatographic modes of two different separation mechanisms,has higher peak capacity and higher separation selectivity than single-dimensional liquid chromatography,has been widely used in proteomics studies.But,the separation of intact proteins and polypeptides from snake venom by 2D-LC has not been reported.Therefore,This study aims to establish an online comprehensive two-dimensional liquid chromatography(LC×LC)system for the separation of intact proteins and polypeptides in snake venom.The first chapter comprenhensively reviewed current separation methods widely used in snake venom proteomics and compared their advantages and disadvantages.Afterwards,the related concepts and construction standards of two-dimensional liquid chromatography were first inroduced,and then its application in proteomics and related modulation methods of LC×LC with different modes were briefly summarized.On this basis,the research outline and innovations of this thesis were put forward.In the second chapter,the effects of different specifications and different stationary phases on the separation of snake venom were investigated using Deinagkistrodon acutus venom as test sample.Two separation modes,i.e.reversed phase liquid chromatography(RPLC)and size exclusion chromatography(SEC)were studied and their optimal chromatographic specifications of were obtained,respectively.The results show that the peak number obtained by RPLC is almost 4～10 times more than that of SEC.The established RPLC method in this study has better separtion performance than literature reported RPLC method(around 1 time more peak numbers),while no signicanct improve of the SEC method in comparasion with literatue.The third chapter constructed a SEC×RPLC system suitable for snake venom based on the above optimized liquid chromatography conditions sample loop modulator was empolyed for the construction of the systme.Moreover,the feasibility of the system was varified using four snake venoms.Taking Deinagkistrodon acutus venom as test sample,the effects of detector sampling frequency,the second dimensional(2D)separation temperature,2D flow rate,2D sampling time and other conditions of the SEC×RPLC system were systematically investigated.As showing by the results,the SEC×RPLC system has better separation perfomance than that of SEC,which demonstrates that the two-dimensional liquid chromatography combined with two orthogonal separation mechanisms has potential in the separation of snake venom.In order to further improve the separation selectivity of LC×LC system,a RPLC×RPLC system was constructed in the fourth chapter by using different acidic and alkaline conditions for the first dimensional(~1D)and ~2D.The effects of ~2D full gradient and parallel gradient on the separation performance of snake venom were compared and optimized.Finally,the feasibility of the RPLC×RPLC system was further confirmed by the separation of Bungarus multicinctus venom.The results show that the parallel gradient more is more suitable for snake venom separation than the ~2D full gradient mode.The results showed that the RPLC×RPLC system has better spearation perfomacne than the SEC×RPLC systme with 57 more chromatographic peaks for Deinagkistrodon acutus venom.It could play a better role in proteomics research and active component screening of snake venoms.The fifth chapter first systematically summarized the results and shortcomings of this research,and then discussed the prospects of the two-dimensional liquid chromatography in snake venomics study. |
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