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Proteins in olive oil have been scarcely investigated probably due to the difficulty of working with such a lipidic matrix and the dramatically low abundance of proteins in this biological material. Additionally, this scarce information has generated contradictory results, thus requiring further investigations. This work treats this subject from a comprehensive point of view and proposes the use of different analytical approaches to delve into the characterization and identification of proteins in olive oil. Different extraction methodologies, including capture via combinational hexapeptide ligand libraries (CPLLs), were tried. A sequence of methodologies, starting with off-gel isoelectric focusing (IEF) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or high-performance liquid chromatography (HPLC) using an ultraperformance liquid chromatography (UPLC) column, was applied to profile proteins from olive seed, pulp, and oil. Besides this, and for the first time, a tentative identification of oil proteins by mass spectrometry has been attempted.

The proteome of untreated white wines (a Recioto made with Garganega grapes from the Veneto region) was explored in depth via capture with combinatorial peptide ligand libraries (CPLL) at four different pH values: pH 2.2, 3.8, 7.2, and 9.3. The combined data on the discoveries in the four CPLL eluates, as well as in the collected bottle sediment, allowed the identification of 106 unique gene products belonging to Vitis vinifera, as well as of an additional 11 proteins released by the S. cerevisiae used in the fermentation process. Among the residual grape proteins detected in the Recioto wine, ca. 30% were categorized as medium to high-abundance species, vs 70% low-abundance ones. The detection of so many low-abundance species suggests that proteomic (coupled to peptidomic) data might be used for typing high-quality products (grand crus) to assess their genuineness and protect them from fraudulent imitations.

The hemolymph of Limulus polyphemus, a very ancient marine arthropod dating back to ca. 440 million years, has been explored in depth via capture by combinatorial peptide ligand libraries. Whereas barely a dozen proteins had been known up to the present, we have increased this number by more than 1 order of magnitude, up to 160 unique gene products, identified via the dbEST_limulus as well as via comparison with the other members of the Chelicerata subphylum to which Limulus belongs, namely, scorpions, ticks, mites, and spiders. Yet we have sequences of many other peptides, suggesting the presence of at least one more order of magnitude of species (1000 and more), that could not be identified as such sequences have no counterparts in present databases. This further reinforces the notion that these could be ancestral proteins, scarcely represented in present times. These data might represent the true birth of paleo-proteomics.

The use of combinatorial peptide ligand libraries, containing hexapeptides terminating with a primary amine, or modified with a terminal carboxyl group, allowed discovering and identifying a large number of previously unreported proteins in cow's whey. Whereas comprehensive whey protein lists progressively increased in the last 6 years from 17 unique gene products to more than 100, our findings have considerably expanded this list to a total of 149 unique protein species, of which 100 were not described in previous proteomics studies. As an additional interesting result, a polymorphic alkaline protein was observed with a strong positive signal when blotted from an isoelectric focusing separation in gel and tested with sera of allergic patients. This polymorphic protein, found only after treatment with the peptide library, was identified as an immunoglobulin (Ig), a minor allergen that had been largely amplified. The list of cow's whey components here reported is by far the most comprehensive at present and could serve as a starting point for the functional characterization of low-abundance proteins possibly having novel pharmaceutical, diagnostic, and biomedical applications.