Proteomic and immunological characterization of a new food allergen from hazelnut (Corylus avellana).

Hazelnuts (Corylus avellana) are one of the most common sources of life-long IgE-mediated food allergies. In this study, we investigated the IgE-reactivity pattern of children with hazelnut allergy (N=15) from Regione Campania, located in Southern Italy, and addressed proteomic strategies for characterizing IgE-binding proteins. For all of the patients (15/15), the predominant IgE-reactive component was a minor ~55kDa protein not previously described. Similar to the hazelnut 11S globulin Cor a 9 allergen, the immunoreactive protein consisted of two subunits linked via a disulfide bridge. In contrast to Cor a 9, only the 20.7kDa alkaline subunit exhibited IgE-affinity. The immunogenic subunit was purified by a two-step chromatographic procedure, but peptide mass fingerprinting was unsuccessful in identifying it, due to the incompleteness of the annotated hazelnut genome. Several tryptic peptides were de novo sequenced by tandem mass spectrometry and showed a high degree of homology with the 11S globulin storage proteins from other seeds, some of which have already been reported as food allergens. The structural characterization suggests that the new putative allergen is a divergent isoform of the hazelnut 11S globulin. These results provide a new platform for developing innovative diagnostic and therapeutic intervention plans. BIOLOGICAL SIGNIFICANCE:Over the years, at least five proteins have been reported as potential food hazelnut allergens. The predominance of specific allergens appears to be strictly related to the geographical origin of the allergic subjects. The complex patterns of the IgE-reactivity of hazelnut storage proteins result in a poor diagnostic and prognostic accuracy. In the perspective of a component-resolved "molecular approach" to the hazelnut allergy we investigated the immune-reactivity patterns to hazelnuts of 15 patients (14 in the pediatric age range) from Region Campania, located in Southern Italy. For all the patients the predominant IgE-reactive component was a minor ~55kDa protein not previously annotated in either protein or genomic databases. The putative allergen was isolated, partially characterized by MS/MS de novo sequencing and appears to be an isoallergen of the hazelnut 11S globulin Cor a 9. Like this latter, the immunoreactive protein consisted of two subunits linked via a disulfide. In contrast to Cor a 9, only the 20.7kDa alkaline subunit exhibited IgE-affinity, in analogy to 11S allergens from other seeds (pistachio, cashew, soybean). We believe that the application of combined immunochemical and proteomic strategies to characterize the new food allergen could be of interest for the readers of Journal of Proteomics. In addition, the results of this study have functional worth in providing a new platform to plan innovative diagnostic and therapeutic intervention approaches to treat hazelnut allergy

A chromatographic and immunoprofiling approach to optimising workflows for extraction of gluten proteins from flour

Ingestion of gluten proteins from wheat, and related prolamin proteins from barley, rye, and oats, can cause adverse reactions in individuals with coeliac disease and IgE-mediated allergies. As there is currently no cure for these conditions, patients must practice avoidance of gluten-containing foods. In order to support patients in making safe food choices, foods making a “gluten-free” claim must contain no more than 20 mg/Kg of gluten. Mass spectrometry methods have the potential to provide an alternative method for confirmatory analysis of gluten that is complementary to analysis currently undertaken by immunoassay. As part of the development of such methodology the effectiveness of two different extraction procedures was investigated using wholemeal wheat flour before and after defatting with water-saturated butan-1-ol. A single step extraction with 50 % (v/v) propan-2-ol containing 2 M urea and reducing agent (buffer 1) was compared with a two-step extraction using 60 % (v/v) aqueous ethanol (buffer 2) followed by re-extraction of the pellet using buffer 1, using either wheel mixing under ambient conditions (19 ◦C) or sonication at 60 ◦C. The procedures were compared based on total protein extraction efficiency and the composition of the extracts determined using a combination of HPLC, SDSPAGE and immunoblotting with a panel of four gluten-specific monoclonal antibodies. Defatting generally had a detrimental effect on extraction efficiency and sonication at 60 ◦C only improved extraction efficiency with buffer 2. Although the single-step and two-step procedures were equally effective at extracting protein from the samples, analysis of extracts showed that the two-step method gave a more complete extraction of gluten proteins. Future studies will compare the effectiveness of these procedures when applied in the sample workflows for mass spectrometry based methods for determination of gluten in food

Hen's egg white database - FASTA format

Full length protein sequences in FASTA format of hen's egg white allergens - including ovomucoid (3 variants), ovalbumin (5 variants), Ovotransferrin (6 variants) and lysozyme (7 variants)

tree nut database - FASTA format

Protein sequences in FASTA format of hazelnut, almond and walnut foor allergen

cow's milk database - FASTA format

Full length protein sequences in FASTA format of cow's milk allergens - including α S1 casein (9 natural variants), α S2 casein (3 natural variants), β casein (13 natural variants), κ casein (13 natural variants), β lactoglobulin (11 natural variants), α lactalbumin (2 natural variants), Serum albumin (8 variants

cow's milk database - FASTA format

Full length protein sequences in FASTA format of cow's milk allergens - including α S1 casein (9 natural variants), α S2 casein (3 natural variants), β casein (13 natural variants), κ casein (13 natural variants), β lactoglobulin (11 natural variants), α lactalbumin (2 natural variants), Serum albumin (8 variants

Hen's egg white database - FASTA format

Full length protein sequences in FASTA format of hen's egg white allergens - including ovomucoid (3 variants), ovalbumin (5 variants), Ovotransferrin (6 variants) and lysozyme (7 variants)

tree nut database - FASTA format

Protein sequences in FASTA format of hazelnut, almond and walnut foor allergen