PROTEIC PROFILE OF THE OLIVE POLLEN BY MALDI TOF/TOF

The term allergenomics has been recently proposed for a rapid and comprehensive analysis of protein allergen candidates by applying a proteomic strategy. The profile of protein extracted from olive pollen might also provide clues for specific cultivar differentiation. In fact, the structure which surround the pollen contains numerous proteins, that vary between plant species. Cultivars and probably local variety or subvarieties of olive trees show special features that depend on their adaptation to the environment or ecotype. The ecoenvironment and crop management are factors able to induce qualitative changes. The aim of this work is to determine a protein profile of the whole extract of the olive pollen and protein identification without preventive chromatography or two dimensional gel separation. Whole pollen proteins of mediterranean cultivars (1-8), obtained by extraction from ammonium bicarbonate, were chemically fractionated in three portions by successively extraction of denaturated protein pellet using solutions at different pH. 1 μL of each fraction was directly analyzed by linear MALDI using α-CHC as matrix. The fraction 7b was digested with trypsin and the resulting peptides mixtures were analyzed by MALDI TOF/TOF. The sequence of the most abundant peptides was confirmed by MS/MS experiments. The mass spectra were acquired on a 4700 proteomics analyzer mass spectrometer in reflectron mode, with 400-ns delayed extraction, averaging 2000 laser shots with a mass accuracy of 10 ppm. The MS/MS experiments were acquired at the collision energy of 1 kV. The pollens from the following mediterranean cultivars Ottobratica (1), Carolea (2), Dolce di Rossano (3), Cassanese (4), Coratina (5), Nocellara del Belice (6), Villacidro (7) and Sinopolese (8) were analyzed. Linear MALDI spectra of the three hydro soluble fraction (a, b, and c) showed ion peaks corresponding to the most common allergenic proteins ole e 1, Poc3_Oleeu and several others polipetides in the range of 6-9 kDa. MALDI spectra of first hydro soluble fraction of (2-8) showed similar protein expression; while the second fraction is very different for each cultivar. In particular, MALDI spectrum of 7b is characterized by the presence of [Ole e 1]2+, [Ole e 1]+, [2Ole e 1]+ at m/z 8896.68, 17810.05 and 17810.05, respectively; the MS information suggested that this fraction contain different alleles of ole e 1. Therefore, 100 μl of this fraction was digested with trypsin and submitted to MALDI reflectron analysis. The sequence specific peptites at m/z 1793.29, 1601.21, 1267.17, 1787.27 and 1039.19 corresponding respectively to AGFITELSEFIPGASVR, ENGDVTFTEIGYTR, AEGLYSMLVER, DCNEIPTEGWAKPSLK and TVNPLGFYK were identified by database searching. The sequence of this peptides was confirmed by MS/MS experiments. Otherwise, the fraction 1b showed four peaks at m/z 9796.75, 9869.78, 9977.54 and 10047.80 corresponding to two glycosilated alleles of Poc3_Oleeu. The preliminary data suggest that the same protein could be expressed at different extent as a function of a specific cultivar. Moreover, the different examined cultivars show, in some cases, a different protein profile which may be used for their identification.