Allergenic Composition of Polymerized Allergen Extracts of Betula Verrucosa, Dermatophagoides Pteronyssinus and Phleum Pratense

Background: Allergoids have been successfully used in the treatment of respiratory allergic diseases. They are modified allergen extracts that allow the administration of high allergen doses, due to their reduced IgE binding capacity.They maintain allergen-specific T-cell recognition. Since they are native allergen extracts that have been polymerized with glutaraldehyde, identification of the allergenic molecules requires more complicated methods. The aim of the study was to determine the qualitative composition of different polymerized extracts and investigate the presence of defined allergenic molecules using Mass spectrometry.

Methods: Proteomic analysis was carried out at the Proteomics Facility of the Hospital Nacional de Parapléjicos (Toledo, Spain). After reduction and alkylation, proteins were digested with trypsin and the resulting peptides were cleaned using C18 SpinTips Sample Prep Kit; peptides were separated on an Ultimate^™ nano-LC system using a Monolithic C18 column in combination with a precolumn for salt removal. Fractionation of the peptides was performed with a Probot^™ microfraction collector and MS and MS/MS analysis of offline spotted peptide samples were performed using the Applied Biosystems 4800 plus MALDI TOF/TOF Analyzer mass spectrometer. ProteinPilot^TM Software V 2.0.1 and the Paragon algorithm were used for the identification of the proteins. Each MS/MS spectrum was searched against the SwissProt 2010_10 database, Uniprot-Viridiplantae database and Uniprot_Betula database.

Results: Analysis of the peptides revealed the presence of native allergens in the polymerized extracts: Der p 1, Der p 2, Der p 3, Der p 8 and Der p 11 in D. pteronyssinus; Bet v 2, Bet v 6, Bet v 7 and several Bet v 1 isoforms in B. verrucosa and Phl p 1, Phl p 3, Phl p 5, Phl p 11 and Phl p 12 in P. pratense allergoids. In all cases, potential allergenic proteins were als identified, including ubiquitin, actin, Eenolase, fructose-bisphosphate aldolase, luminal-binding protein (Heat shock protein 70), calmodulin, among others.

Conclusions: The characterization of the allergenic composition of allergoids is possible using MS/MS analysis. The analysis confirms the presence of native allergens in the allergoids. Mayor allergens are preserved during polymerization.