The Citrus clementina putative allergens: From proteomic analysis to structural features

Several allergens have been identified and characterized in the genus Citrus, which belongs to the germin-like proteins (GPLs), profilins, and non-specific lipid transfer proteins (nsLTPs). In this work, in silico sequence analysis, protein purification, mass spectrometry identification, and the spectral counting method were integrated to identify new putative allergens of Citrus clementina and their expression level in the fruit peel. The in silico analysis revealed fifteen new sequences belonging to GLPs (Cit cl 1), and two more belonging to nsLTPs (Cit cl 3). No other new sequences were found as regards profilins (Cit cl 2). Each putative allergen from fruit peel was obtained using different protein extraction methods, and the protein sequences of the putative allergens were identified by means of LTQ-Orbitrap XL mass spectrometer. The spectral counting strategy revealed that Cit cl 1 had a higher expression level than Cit cl 2 and Cit cl 3. To predict the quaternary structure and deduced function of Cit cl 1, its primary sequence was used as a template to search a homologous protein structure in the RCSB PDB Database, getting high correspondence with the oxalate oxidase protein in barley.