1. THE PROTEOME OF OLIVE SEEDS AND THE INVISIBLE PROTEOME OF OLIVE OILS AS DETECTED VIA COMBINATIONAL PEPTIDE LIGAND LIBRARY CAPTURE Clara Esteve a, Alfonsina D’Amato b, M. Luisa Marina a, M. Concepción García a, Pier Giorgio Righetti b a Department of Analytical Chemistry, University of Alcalá. Alcalá de Henares (Madrid), Spain b Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano. Milan, Italy ♠ Olive fruit and olive oil proteins have been scarcely investigated, despite their highly informative value and role in food stability and allergenicity. ♠ There is a high difficulty in extracting proteins from vegetable matrices rich in lipids. The use of combinational peptide ligand libraries (CPLLs) at pH 7.4 and 2.2 has been employed in the extraction protocols. The main objective of this work is to establish a suitable method for the isolation of proteins from olive seed and olive oil based on the application of the most improved CPLL technology and for the detection of olive fruit and olive oil proteome. Protein extraction Experimental conditions ♠ Chromatographic conditions - Column: Acclaim PepMap 100 C 18 (20 mm x 100 µm) - Mobile phases: A (0.1% (v/v) FA, 2% (v/v) AcN in water) B (0.1% (v/v) FA, 2% (v/v) water in AcN) - Gradient: 4% to 40 % phase A in 30 min. - Flow rate: 300 µL/min - Injected volume: 8 µL ♠ ESI-LIT - Equipment: LTQ XL (Thermo Scientific) MS/MS Analysis Software MASCOT Search Engine (Version 2.3.01) Database: Uniprot_viridiplantae (30264 residues, 184678199 sequences) Precipitate redisolution in 4% SDS 25 mM DTT, 100 ºC +400 mL 25% TCA/Acetone Precipitation MeOH/CH 3 Cl Olive oil (400 mL) SDS-PAGE-Silver staining M. C. García thanks financial support from project CTQ2009-11252 (Ministry of Science and Innovation, Spain). M. L. Marina and M. C. García thank financial support from project S-2009/AGR-1464 (Comunidad Autónoma of Madrid, Spain). C. Esteve thanks the University of Alcalá for her pre-doctoral and mobility grants and additionally FEBS (Federation of European Biochemical Societies) for a short-term fellowship for a stage at the Politecnico di Milano. ♠ It has been possible to identify 61 unique gene products in the olive seed, representing an important increment in the exploration of seed species. ♠ A high number of isoforms of histones and globulin seed storage proteins, and an oleosin of 17,2 kDa. ♠ It has been confirmed that CPLL treatment permits identifying a much larger number of species than in control, untreated sample, even in such recalcitrant tissues as plant material. ♠ For olive oil, we could assess the presence of very fine bands in SDS-PAGE gels, centered in the 10-30 kDa regions, whose identification is now in progress. Such species are found only in trace amounts, though, and can only be visualized via silver staining, thus their identification might not represent an easy task. Olive seed SDS-PAGE-Coomassie blue Tris-HCl (pH 7.4), NaCl, EDTA, DTT, SDS, protease inhibitor cocktail CPLLs (pH 7.4) Urea, thiourea, CHAPS, protease inhibitor cocktail Tris-HCl (pH 7.4), NaCl, CHAPS, protease inhibitor cocktail CPLLs (pH 7.4) CPLLs (pH 2.2) Proteins SDS-PAGE separation, digestion, and analysis Trypsin digestion nLC-MS/MS (LTQ-XL) Protein identification INTRODUCTIONOBJECTIVE EXPERIMENTAL RESULTS CONCLUSIONS ACKNOWLEDMENTS Olive seedOlive oil Coomassie Blue staining Silver staining Venn Diagram Gene Ontology The various captures with CPLLs permitted the additional identification of 31 unique gene products It demonstrates the role of seed in containing genetic material for plant development. OLIVE SEED OLIVE OIL FUTURE ANALYSIS IN A MORE SENSITIVE EQUIPMENT