Exploring Of Snakebites Identification Technique By Means Of Proteomics

Snakebite is common in tropical and subtropical regions. In rural areas of Africa, Asia, Oceania and Latin America, snakebite is particularly prominent public health problem. A recent study show: Worldwide, the annual injured number due to snakebite was more than 500 million, causing 25,000-125,000 deaths. China snakebite incidence mainly occurs in the southern provinces, more common in summer and autumn. It was reported that the number of snakebites each year was more than 100,000, of which nearly 10,000 people will die. And, in recent years, the incidence of snakebite increased year by year because environmental changes, feeding snakes and field operations, etc.In forensic work, we often meet cases suspected of snake venom poisoning. At present, the identification of these cases is mainly dependent on a general medical examination, observing the teeth marks, symptoms and biochemical routine examination, and the key is the teeth marks. However, we also often encountered some cases without obvious teeth marks, thus we had to identify snakebite through the symptoms and biochemical indicators. This make the possibility of erroneous judgment very large. Therefore, it is imperative to develop new research methods to address this topic.In this study, we used proteomics technology based on liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS) to research some common snake venom within China, including Naja atra, Deinagkistrodon acutus, Bungarus multicinctus and Bungarus fasciatus. Furthermore, we compared the similarities and differences in different types of venom, and research the changes of rat endogenous proteins caused by venom so that obtain venom poisoning biomarkers, and to provide a new method for the identification of snakebite cases. The main content is divided into the following three sections.SectionⅠProteomics study of four kinds of snake venomIn order to understand the proteins characteristics of different venom, we digested Naja atra venom, Deinagkistrodon acutus venom, Bungarus multicinctus venom and Bungarus fasciatus venom into peptides, then detected these peptides with LC-MS/MS. The data was analyzed using label-free quantification technology(LFQ), so as to obtain the protein abundance. From the four kinds of venom, we found some high abundance proteins. In Deinagkistrodon acutus venom, metalloproteinases, C-type lectins and antithrombin showed a similar abundance, were 25.4%, 24.6% and 23.6%, respectively. In Naja atra venom, cardiotoxins showed the highest abundance(77.1%). In Bungarus multicinctus venom, the abundances of beta-bungaratoxin and short neurotoxin homolog NTL4 were 21.1% and 46.6%, respectively. In Bungarus fasciatus venom, the abundance of acetylcholinesterase was 39.8%. The results show that different venoms have significant differences on protein composition and abundance, and these differences can serve as the foundation of snakebite identification.Section II Serum protein change caused by venoms and poisoning durationIn order to explore the serum protein change caused by venom, we injected the aforementioned four kinds of venom into the SD rats, respectively, and collected rat serum at a predetermined time point. Rat serum proteins were detected using LC-MS/MS after a series treatment, then the identification and quantification of serum proteins were carried out using Peaks Studio software. Quantitative information was clustered and analyzed so as to look for some proteins whose abundance were changed gradually. Subsequently, these proteins owning significant change were performed IPA, including downstream functional effects analysis, pathway analysis, regulatory trends, protein-protein interaction network analysis. Furthermore, Peaks Studio’s quantitative results was screened according to the ratio <0.7 or> 1.33(the experimental group protein intensity divided by the control group protein intensity) to obtain the significantly different serum proteins in each group. The results showed that total 83 proteins abundance were changed in rat serum of Deinagkistrodon acutus venom group, and mainly distributed in some protein family associating with blood clotting and cytoskeletal such as Fibrinogen, Coagulation factor, Tropomyosin, Tubulin, Myosin. Naja atra venom groups were divided into the high-dose group and the low-dose group, there were 47 and 28 protein concentration changed, respectively. In Bungarus multicinctus venom group, less proteins were changed, and these proteins were found to associated with the neural pathways. These analysis results indicate that various snake venom can lead to different endogenous proteins are changed. This maybe help us to identify different snakebite. Further, these significantly different proteins was carried out systematic literature survey. Then, we chose Retinol-binding protein 4, L-selectin, Troponin I, Protein Z-dependent protease inhibitor as candidate biomarkers of Naja atra venom poisoning; Fibrinogen as a candidate biomarker of Deinagkistrodon acutus venom poisoning; EH domain-containing protein as a candidate biomarker of Bungarus multicinctus venom poisoning.Section III Muscle tissue protein change caused by snake venomIn order to explore the muscle tissue protein change caused by venom, we injected the four kinds of venom into the SD rats, respectively. After 4 hour, we killed the rats humanity and collected their muscle tissue of injection site. The proteins from muscle tissue were detected using LC-MS/MS after a series treatment, then the identification and quantification of muscle proteins were performed by Peaks Studio software. Quantitative information was clustered and analyzed in order to find some proteins whose concentration occurred change. Furthermore, these proteins owning significant change were performed IPA, including functional effects analysis, pathway analysis, protein-protein interaction network analysis. And, Peaks Studio’s quantitative results was also screened according to the ratio <0.7 or > 1.33(the experimental group protein intensity divided by the control group protein intensity) to obtain the significantly different muscle proteins in each group. Our data indicate that muscle proteins are significantly different after injected venom, and different venom can cause the different protein change. An interesting finding is MPPA(one of venom components) is significantly up-regulated in wound muscle of all five groups rat, it might serve as venom poisoning exogenous marker. In Deinagkistrodon acutus venom group, HBA / HBB, LEG5, FGA were significantly up-regulated relative to the control group and the other groups, they might be considered as candidate biomarkers indicating Deinagkistrodon acutus venom poisoning. In Bungarus multicinctus venom group, GC, and GELS were significantly up-regulated relative to the control group, while IPKA, ACSL1, DOPD and ACADV were down-regulated relative to the control group, the 6 proteins is expected to become candidate biomarkers of Bungarus multicinctus venom poisoning. In Bungarus fasciatus venom group, the abundance of FGA and FGB were up-regulated more than two times, and other endogenous proteins such as PLMN, TF, ALB, C9 and APOH also have significantly higher expression relative to other 4 experiments groups. Thus, these proteins may be considered as candidate biomarkers of Bungarus fasciatus venom poisoning. In Naja atra venom group, GELS, HSP71, ATN2 and SYNPO were significantly differentially expressed, can be used as a sign of Naja atra venom poisoning.ConclusionThis is first systematically study on Naja atra venom, Deinagkistrodon acutus venom, Bungarus multicinctus venom and Bungarus fasciatus venom(common venom in China). In this study, we revealed proteomics characteristic of these 4 kinds of venom using LC-MS/MS, disclosed the changes of serum proteins and muscle proteins caused by venom, and obtained the biological pathways and protein regulatory networks of venom. We also selected some proteins as candidate biomarkers of venom poisoning through rigorous screening endogenous protein concentration changes caused by venom poisoning. These research results provide a new approach and new idea for forensic identification of snakebite cases.