Identification Of Major Protobothrops Mucrosquamatus Venom Allergens Using Immunoproteomics And Mass Spectrometric Approaches

BackgroundSnake venoms are complex mixtures of proteins and non proteins components. They exhibited various biological and pharmacological activities. Though they can poison the human and animals, they are rich resources for drug candidates. Many drugs derived from snake venoms have been used in clinical practice or in clinical study stage. However, as allogeneic proteins, snake venom derived drugs have found to cause allergenic reactions with symptoms ranging from skin maculopapule to life-threatening anaphylaxis and dead. Snake venoms themselves also caused serious allergenic reactions in specific population who are close relationship with snake or snake venom in their daily life.ObjectTaking Protobothrops mucrosquamatus (Pmu) snake venom collected in Hunan province as example, we identified the allergens from this snake venom by comprehensive methods including immunoproteomics and mass spectrometric analysis.MethodsImmunoproteomics combined with mass spectrometric analysis was used to identify proteins of Pmu venom responsible for Pmu-specific IgE immunoreactivity, using sera from Pmu-sensitized patient. The venom protein components were separated by two-dimensional electrophoresis (isoelectric focusing as first dimension and SDS-PAGE as the second dimension). Then the gel was transferred onto PVDF membrane. The transferred PVDF membranes were blocked for overnight and incubated with individual sera from Pmu-allergic patients and nonatopic control subjects, respectively. After washing in TBST, the membranes were incubated with monoclonal anti-human IgE-Abs. The PVDF membranes were extensively washed in TBST, and detected with an enhanced chemiluminescence kit. The protein spots matched immunoblotting signal were manually picked from the coommassie-stained gels run, digested with trypsin and analyzed by means of multidimensional liquid chromatography-ion trap mass spectrometry.ResultsWater/salt-soluble snake venom proteins of the Pmu were separated by means of two-dimensional gel electrophoresis with immobilized pH gradients (IPGs) and SDS-PAGE and83different proteins were detected. The two-dimensional is a powerful protein separation technique, with high resolution and large-scale analysis. Even isoallergens with small differences in pI or molecular mass corresponding to sequence isoforms or different posttranslationally modified protein variants could be resolved and distinguished from each other. The relative molecular mass (Mr) of approximately90%total proteins were between15-45kDa. The isoelectric points (pI) for approximately72.29%of total proteins were between4.0-7.0.Allergens have been identified by immunoblotting incubating with the sera of the7symptomatic patients following two-dimensional electrophoresis. All patients were sensitized to one or more allergen protein and9IgE-binding spots were detected. Each patient showed an individual IgE-binding pattern with4to9different allergen spots. The relative molecular mass (Mr) and the isoelectric points (pI) of total proteins were between30-40kDa/5.20-7.84. The most abundant IgE-binding spots were numbers6,8and9, with100%of reactions. None of IgE negative serums shown IgE-binding spots in the control experiments.Nine Coomassie-stained protein spots were analyzed after tryptic digestion by means of multidimensional liquid chromatography-ion trap mass spectrometry. The result of MS/MS analysis showed that they belong to5different protein classification including mucofirase3, serpentokallikrein-2, Plasminogen activator Haly-PA, H2proteinase and metalloproteinase PMMP-3. More than one of the allergen spots was detected as a full-length protein. Spot1is the best sequence coverage rate, as high as66.15%, and spot9is the least.ConclusionsIn conclusion, our results demonstrate the inter-individual variation of Pmu sensitization in snake venom allergy and the applicability of2-dimensional electrophoresis with IgE immunoblotting and mass spectrometric to identify allergens. The reason for the strong diversity of reactions among snake venom allergy is not known. The immunoproteomic approach applied to the analysis of Pmu venom protein laid a solid foundation to allergens’proteomics and molecular biology basic research of snake venom, as well as provided a theoretical basis to the specific diagnosis and treatment of Pmu allergic diseases. These findings will undoubtedly contribute to a deeper understanding of snake venom and the design of new drugs in search of leading compounds.