Characterization by 2DE and LC-MS of hazelnut oleosins allergy in Italian pediatric patients

Very little is known about allergic reactions caused by oleosins (Cor a 12 and 13) in pediatric patients affected by hazelnut allergy. The main reason is due to the absence of oleosin because of their hydrophobic nature from the protein extracts used in diagnostic tests (1). The aim of this work was: (i) the investigation of false negative results obtained by hazelnut allergy diagnostic test and (ii) the evaluation of how hazelnut roasting could modify oleosin allergenicity. A protocol for oil bodies associated proteins extraction was optimized and SDS-PAGE under reducing and non-reducing condition was performed. The electrophoretic profile showed two most abundant bands identified by LC-MS/MS analysis as Cor a 12 and 13 in the raw sample, while additional minor bands corresponding to Cor a 9 and Cor a 11 were found in roasted samples, probably due to their adhesion to the oil body after roasting. The IgE binding pattern against the oil body protein extracts was determined by immunoblotting with 30 hazelnut allergic patients sera. The immunoblotting results showed that 85% of patients have IgEs versus at least one oleosin in raw hazelnut and 90% in roasted hazelnut. In order to verify that the patients reactivity could be ascribed only to oleosins a 2DE of oil body protein extract was performed. Separating proteins according to pI by 2DE, allowed to discriminate oleosin, characterized by alkaline pI (10.54 and 9.98, respectively) from the other co-migrating allergens (Cor a 9 and Cor a 11). The 2DE-immunoblotting showed a strong reactivity only in the very alkaline spots containing exclusively oleosins. These results demonstrate that oleosins are high-frequency recognized allergens in pediatric Italian patients and they maintain their allergenicity also after protein modification induced by roasting.