D2.198 - Allergen identification and characterization of lysine modification in monomeric allergoid of Olea europea pollen extract

Carbamylation of allergens through potassium cyanate reaction (KCNO) substitutes the ɛ-amino group of lysine residues of proteins with ureido groups without changing molecular size. Such an approach has been used to develop AIT vaccines with reduced allergenicity profile (monomeric allergoids). This modification significantly reduces the IgE-binding capacity of the extract but also prevents the detection of allergenic molecules by monoclonal or polyclonal antibodies in the allergoid extract using conventional immunological methods. The question of analyzing proteins identification and determining the lysines modification in a sample of Olea europea carbamyled extract was here addressed by proteomic approach. 

Linearized and alkylated native and KCNO-modified allergen extracts of Olea europea were digested by incubation with proteolytic enzymes (trypsin and proteinase K). The obtained peptides were comparatively analyzed by nanoLC-MS/MS-based sequencing. The resulting chromatographic run files were analyzed using Proteome Discoverer 2.3 software, using the Olea europea proteome database on Uniprot (79,665 proteins).

Peptide sequencing identified 27,955 proteins and confirmed the presence of the important allergens and their isoforms annotated in WHO/IUIS Database. Thus, allergens of groups 1, 2, 3, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 were detected in native and chemical modified Olea pollen extracts. Furthermore, several carbamylated lysines positioned in the context of  clinically relevant molecules (i.e. Ole e 1, Ole e 7, Ole e 9, associated with disease severity, asthma diagnosis, or systemic reactions to immunotherapy) were identified in the modified extract.

nanoLC-MS/MS-based sequence analysis represents a useful method to evaluate the number and position of modified lysine residues. KCNO-modification of Olea europea allergen extract appears to not impair the presence of clinically relevant allergenic molecules and determines a significant carbamylation of lysines in critical allergenic molecules.