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Dealing with the identification of protein species in ancient amphorae

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Abstract

This manuscript deals with the identification of protein residues in amphorae, including particularly identification of protein species. The work described was performed on fishes, the anchovy (Engraulis encrasicolus) and bonito (Sarda sarda) species frequently found in the Mediterranean area. Based on proteomic techniques, the analytical strategy was adapted to analysis of protein residues from tiny ceramic fragments. The major difficulty was to extract proteins and limit their hydrolysis during the sample preparation; consequently, multiple soft extraction techniques were evaluated. The most valuable results were obtained using a solution containing high amounts of denaturing agents, urea and thiourea, reducing agent, dithiothreitol, and detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The analysis using nano liquid chromatography–nano electrospray ionization double quadrupole time-of-flight mass spectrometry resulted in the identification of up to 200 proteins for the anchovy and bonito species, among which 73 peptides were found to be fish-specific. Because bonito and anchovy species are not documented and fully sequenced in genomic databases, the preliminary protein identification was realized via sequence homology to other fish sequenced species. Amino acid substitutions of peptides were assigned on the basis of the interpretation of tandem mass spectrometry spectra using de novo sequencing; these peptides, not reported up to now in databases, constitute species-specific markers. The method developed was finally applied to an archaeological sample replica impregnated with a mixture of fish tissue from both species; this experiment successfully led to the identification of 17 fish proteins, including 33 fish-specific peptides. This work shows that the analytical method developed has great potential for the identification of protein species in complex archaeological samples.

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Acknowledgements

This work was funded by the Agence Nationale de la Recherche ANR-08-JCJC-0082-01. The mass spectrometry facilities used for this study are funded by the European Community (FEDER), the Région Nord-Pas de Calais (France), the IBISA (Infrastrutures en Biologie Santé et Agronomie) network, the CNRS, and the Université Lille 1 Sciences et Technologies.

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Authors and Affiliations

  1. Miniaturisation pour l’Analyse, la Synthèse & la Protéomique (MSAP), USR CNRS 3290, and Protéomique, Modifications Post-traductionnelles et Glycobiologie, IFR 147, Université de Lille 1 Sciences et Technologies, 59655, Villeneuve d’Ascq Cedex, France

    Sophie Dallongeville, Christian Rolando & Caroline Tokarski

  2. SARL Laboratoire Nicolas Garnier, 63270, Vic le Comte, France

    Nicolas Garnier

  3. Facultad de Filosofía y Letras, Depto. Historia, Geografía y Filosofía, Universidad de Cádiz, 11003, Cádiz, Spain

    Dario Bernal Casasola

  4. UMR CNRS 6573, Maison Méditerranéenne des Sciences de l’Homme (MMSH), Centre Camille Jullian, 13094, Aix-en-Provence Cedex, France

    Michel Bonifay

Authors
  1. Sophie Dallongeville
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  2. Nicolas Garnier
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  3. Dario Bernal Casasola
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  4. Michel Bonifay
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  5. Christian Rolando
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  6. Caroline Tokarski
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Corresponding author

Correspondence to Caroline Tokarski.

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Published in the special issue Analytical Chemistry for Cultural Heritage with Guest Editors Rocco Mazzeo, Silvia Prati, and Aldo Roda.

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Dallongeville, S., Garnier, N., Casasola, D.B. et al. Dealing with the identification of protein species in ancient amphorae. Anal Bioanal Chem 399, 3053–3063 (2011). https://doi.org/10.1007/s00216-010-4218-2

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  • DOI: https://doi.org/10.1007/s00216-010-4218-2

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