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For those analyses, we were kindly provided with three types of high-quality oils by the Tenuta Zimarino Masseria Don Vincenzo (Vasto, Chieti, courtesy of Mr. Tieri) namely: Per Liliana “Ascolana Tenera” (monocultivar); Costa dei Trabocchi “Gentile di Chieti” (monocultivar); Don Vincenzo “Colline Teatine”, Vastese DOP.

Clara Esteve1, Alfonsina D'Amato, Pier Giorgio Righetti

Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Proteomics Laboratory, Politecnico di Milano, 20131 Milan, Italy

1Department of Analytical Chemistry, Faculty of Chemistry, University of Alcalá, Spain

It all started, perhaps, in the year 2001 when Hidalgo et al. [1] claimed finding proteins in olive oils, ranging in concentration from 10 to 50 micro-grams per 100 grams of oil. Curiously, though, there was no identification, via the classical tool of mass spectrometry (MS) of any of these presumptive proteins. In addition, all the proteins they claimed having seen amounted to just a single polypeptide chain, of apparent Mr of 4600 Da (but of undisclosed nature). The fact that such an important finding, surely of great interest in the field of olive oil industry, had no follow up in the scientific literature for more that 11 years should have rung a bell.

Notwithstanding that, considering that at least since two years our lab has started an extensive investigation on the proteome content of alcoholic and non-alcoholic beverages, in order to find if they could be classified according to the set of proteins they contained, and if any adulteration could be detected [2-6], we recently decided to visit again this obscure aspect of olive oils, with the following aims:

a) in order to see if enough proteins could be detected so as to classify the various oils according to different cultivars;

b) in order to see if high-quality oils could be distinguished form industrial oils available in supermarket.

As a pre-requisite to that, we first started investigating the proteome content of the olive fruit, on which not much was known up to the present. To that aim, both the proteinaceous content of the seed as well as of the pulp were analyzed, since both compartments could contribute to the presence, if any, of trace proteins in the final olive oil processed. Indeed our work resulted in a very extensive exploration of both proteomes, amounting to the identification of a grand total of 61 proteins in the seed and as many as 231 species in the pulp [7]. Having established these two proteomes, we started investigating the trace proteins in commercial olive oils, as found on the shelves of supermarkets. On the few major olive oils available (Monini, Dante, Carapelli) we could not detect any proteinaceous material. We thus resorted to the analysis of high-quality oils, that had not undergone any industrial refinement process, to ascertain the possible presence of proteins in traces. For those analyses, we were kindly provided with three types of high-quality oils by the Tenuta Zimarino Masseria Don Vincenzo (Vasto, Chieti, courtesy of Mr. Tieri) namely:

  1. Per Liliana "Ascolana Tenera" (monocultivar);
  2. Costa dei Trabocchi "Gentile di Chieti" (monocultivar);
  3. Don Vincenzo "Colline Teatine", Vastese DOP.

A representative label of these three oils is given in Fig. 1

We had to test different extraction protocols in order to obtain reasonable results, but finally we succeed in seeing bands of protein zones in a sodium dodecyl sulphate polyacrylamide gel slab (SDS-PAGE), see Fig. 2. This gel is interesting, since it shows a spectrum of fine bands spanning the Mr region from ca. 12 to 40 kDa, suggesting indeed the presence of quite a few proteins extracted from these types of oils (this gel is representative of all extractions performed in all oils from Masseria don Vincenzo). It also gives us an important clue: considering that the proteins were extracted from 400 grams of oils, and that detection has been performed with silver staining (the most sensitive stain available), likely the trace proteins present should be of the order of tens of micro-grams per litre, i.e. two orders of magnitude lower than that reported in [1].

For the analyses of these presumptive proteins, the gel was segmented into 21 zones (marked by numbers and brackets in Fig. 2), the proteins digested with trypsin, the resulting peptides captured, purified and injected in two mass spectrometer: an Orbitrap (at the Faculty of Pharmacy, Prof. Aldini) and a Velos (at the Fondazione Filarete). Identifications were not easy at all, since the signals was very feeble and there were plenty of traces of any possible contaminant. Finally, after rejecting all spurious spectra and submitting the few surviving ones to stringent statistical tests, we enucleated just three proteins that had survived the threshold test. It should be noted that one of them, histone H4, is highly homologous to a histone that we found in the seed of the olive fruit, which suggest that this protein was transferred to the oil form the proteome of the seed. The other two proteins are homologous to components of the pulp.

Finally, Fig. 3 gives the fragmentation spectrum of one peptide of the histone, giving the determination of the correct sequence of this peptide.


It would appear that only traces of proteins can be found in olive oils, of which at the moment only three have been correctly identified. Surely quite a few more should be present, but ascertaining their IDs might be a difficult task, since such proteins could be very hydrophobic and contain too few Lys and Arg residues, where the trypsin attack occurs. We will thus have to test different enzymes for protein digestion, in the hope of obtaining the correct cuts. The second important information is that it appears that only high quality oils, which have not been subjected to any industrial treatment, contain traces of proteins. Commercial oils commonly found in the supermarkets did not appear to contain any trace proteins (which presumably are removed in the industrial fining process). Thus the present data seem to suggest that the presence of trace proteins might enable consumers and control agencies to distinguish high-quality oils (those that have been prepared by cold pressing in the absence of any subsequent industrial treatment) vs. the industrial ones that have undergone refining processes.


[1] Hidalgo FJ, Alaiz M, Zamora R, Determination of peptides and proteins in fats and oils. Anal Chem. (2001) 73, 698-702.

[2] D'Amato A, Fasoli E, Righetti PG. Harry Belafonte and the secret proteome of coconut milk. J Proteomics (2012) 75, 914-920.

[3] Fasoli E, D'Amato A, Citterio A, Righetti PG. Ginger Rogers? No, Ginger Ale and its invisible proteome. J Proteomics (2012) 75, 1960-1965.

[4] Di Girolamo F, D'Amato A, Righetti PG. Horam nonam exclamavit: sitio. The trace proteome of your daily vinegar. J Proteomics (2011) 75, 718-24.

[5] Fasoli E, D'Amato A, Kravchuk AV, Citterio A, Righetti PG. In-depth proteomic analysis of non-alcoholic beverages with peptide ligand libraries. I: Almond milk and orgeat syrup. J Proteomics (2011) 74, 1080-1090.

[6] D'Amato A, Fasoli E, Kravchuk AV, Righetti PG. Going Nuts for Nuts? The Trace Proteome of a Cola Drink, as Detected via Combinatorial Peptide Ligand Libraries. J Proteome Res. (2011) 10, 2684-2686.

[7] Clara Esteve C, D'Amato A, Marina ML, García MC, Citterio A, Righetti PG. Identification of olive (Olea europaea) seed and pulp proteins by nLC-MS/MS via combinatorial peptide ligand libraries. J Proteomics (2012) 75, 2396-403.

Figure 1 – Label of the monocultivar Ascolana Tenera Oil

Figure 2 – SDS-PAGE analysis of proteins extracted from olive oil "Masseria Don Vincenzo" from Tenuta Zimarino. Mr: molecular mass ladder. The track "Olive oil" has been segmented into 21 zones, whose content has been digested with trypsin and subjected to MS analysis on a Orbitrap.

Figure 3 - MS spectrum of the second peptide of histone R4 (ISGLIYEETR).

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