Analytical and Bioanalytical Chemistry

, Volume 399, Issue 9, pp 3053–3063 | Cite as

Dealing with the identification of protein species in ancient amphorae

  • Sophie Dallongeville
  • Nicolas Garnier
  • Dario Bernal Casasola
  • Michel Bonifay
  • Christian Rolando
  • Caroline Tokarski
Original Paper


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.


Protein species Archaeological ceramics Proteomics Tandem mass spectrometry Amino acid substitution 



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.

Supplementary material

216_2010_4218_MOESM1_ESM.pdf (557 kb)
ESM 1 (PDF 556 kb)
216_2010_4218_MOESM2_ESM.pdf (499 kb)
ESM 2 (PDF 498 kb)
216_2010_4218_MOESM3_ESM.pdf (501 kb)
ESM 3 (PDF 501 kb)


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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Sophie Dallongeville
    • 1
  • Nicolas Garnier
    • 2
  • Dario Bernal Casasola
    • 3
  • Michel Bonifay
    • 4
  • Christian Rolando
    • 1
  • Caroline Tokarski
    • 1
  1. 1.Miniaturisation pour l’Analyse, la Synthèse & la Protéomique (MSAP), USR CNRS 3290, and Protéomique, Modifications Post-traductionnelles et Glycobiologie, IFR 147Université de Lille 1 Sciences et TechnologiesVilleneuve d’Ascq CedexFrance
  2. 2.SARL Laboratoire Nicolas GarnierVic le ComteFrance
  3. 3.Facultad de Filosofía y Letras, Depto. Historia, Geografía y FilosofíaUniversidad de CádizCádizSpain
  4. 4.UMR CNRS 6573, Maison Méditerranéenne des Sciences de l’Homme (MMSH)Centre Camille JullianAix-en-Provence CedexFrance

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