A proteomic approach for the characterization of olives from “Piantone di Mogliano” cultivar.

“Piantone di Mogliano” is an olive cultivar from Marche region, spread mainly in the province of Macerata, with the largest concentration in Mogliano, Macerata and surrounding area, at altitudes close to 600 m on the sea level. Olive drupe mesocarp contains important metabolites such as polyphenols, carotenoids, chlorophylls, sterols, terpenoids and a wide range of volatile compounds which all together influence the quality and aroma of the fruit. Furthermore, in the olive pulp where there are enzymes involved in several metabolic processes and in the development of phenolic and aroma compounds, such as: hydroperoxide lyase (provides the major components of the aroma of virgin olive oil), lipoxygenase (involved in plant response to stress), isopentenyl diphosphate isomerase and polyphenol oxidase (involved in the phenolic metabolism in plants), chitinases (involved in growth and development processes). Therefore, olive fruit development is a combination of biochemical and physiological events that occur under strict genetic control and are influenced by several environmental conditions [1]. The aim of the present work is to achieve a sort of "identity card" which is fundamental for the traceability of olives from “Piantone di Mogliano” cultivar and is an important aid to qualify and classify this food. At this purpose, a proteomic approach based on two-dimensional electrophoresis (2-DE) was used for protein identification in the olive fruit. Since olive fruit is rich in lipids, polyphenols and terpenes which interfere with protein extraction reducing their solubility and changing their properties, it is important to set up an efficient protein extraction method which includes procedures aimed to eliminate the lipid fraction and enhance and concentrate the protein fraction. In this work, a protein extraction protocol based on the modification of already published methods has been developed [2]. Briefly, the frozen olive pulp was ground using liquid nitrogen into a fine powder. The proteins were extracted from the olive powder through a first TCA/acetone extraction procedure, followed by a second phenol extraction procedure. The final olive protein pellet was resuspended in a rehydratation buffer containing urea and detergent and subjected to isolelectric focusing on an immobilized pH gradient 3-10 (Immobiline DryStrip gel, 18cm, GE Healthcare). The second dimension consisted of a 15% SDS-PAGE [3]. After electrophoresis, the proteins were stained with Coomassie blue. The gel was subjected to image analysis (by a PDquest software) and to mass spectrometry for protein identification. As a result, 24 main protein spots were found, each of them analyzed by the PDQuest software for spot quantitation, and determination of pI and molecular weight. The most representative spots were excised from the gel and actually and are under LC-MS/MS analysis for spot identification. Thus, thanks to this protein extraction protocol, it will be possible to identify specific markers aimed to determine the quality and the characteristics of the “Piantone di Mogliano” cultivar, such as proteins associated to the metabolism of fatty acids, phenolic and aroma compounds and also to perform an investigation into the changes that occurs during fruit development and subsequent transformation processes.