title: Dealing with the identification of protein species in ancient amphorae.
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.
authors.affiliation: 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.
authors.forename: Sophie
authors.initials: S
authors.lastname: Dallongeville
authors.forename: Nicolas
authors.initials: N
authors.lastname: Garnier
authors.forename: Dario Bernal
authors.initials: DB
authors.lastname: Casasola
authors.forename: Michel
authors.initials: M
authors.lastname: Bonifay
authors.forename: Christian
authors.initials: C
authors.lastname: Rolando
authors.forename: Caroline
authors.initials: C
authors.lastname: Tokarski
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001106:Archaeology; D001154:Art; D002853:Chromatography, Liquid / Q000379:methods*; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics; D005399:Fishes / Q000235:genetics; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D029941:Fish Proteins; D011506:Proteins