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.

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry combined with multidimensional scaling, binary hierarchical cluster tree and selected diagnostic masses improves species identification of Neolithic keratin sequences from furs of the Tyrolean Iceman Oetzi

“…[24,27,49,50] Other sources of ancient proteins are dental pulps [51] or even contents of ceramics like amphorae. [52] Despite their potential use, hard keratins are an unexploited source of ancient proteins. The keratin-based method used in this study (SIAM = Species Identification of Animals using MALDI-TOF mass spectrometry) was developed for the identification of native feathers, down and hair.…”

Proteomic identification of adhesive on a bone sculpture-inlaid wooden artifact from the Xiaohe Cemetery, Xinjiang, China

“…The technique has been successfully applied in the identification of remains in archaeological pottery (Dallongeville et al, 2011a;Solazzo et al, 2008), binders in the paintings (Fremout et al, 2010;Leo et al, 2009), protein additives in building materials , etc. This work shows the efficiency of proteomic approaches on the identification of the prehistoric adhesive used as inlay material on a staff collected from the Xiaohe Cemetery, Xinjiang, China.…”

Multi-technique chemical characterisation of a 12–13th-century painted Crucifix

“…In particular, MALDI-MS offers a series of advantages such as direct analysis of complex mixtures with reduced sample pretreatment, short analysis times, relatively easy interpretation of results, and high sensitivity, fundamental in the study of paintings with very limited sample amounts. ESI-MS not only allows to identify proteins but also the protein species origin from few micrograms of artwork samples [31,33,35].…”

“…Py-GC/MS is particularly suitable for the analysis of synthetic polymers [12][13][14][15] or polymeric fractions of natural resins [16][17][18][19], whereas MALDI-TOF-MS has recently been shown to be efficient in fast and complete identification of historic paint binders. In fact, since a decade the use of new mass spectrometric techniques based on soft ionization, such as MALDI [20][21][22][23][24][25][26][27] or Electrospray Ionization (ESI) [25][26][27][28][29][30][31][32][33][34][35][36] for investigation of organic materials as present in polychrome artefacts or archaeological finds has significantly increased. In particular, MALDI-MS offers a series of advantages such as direct analysis of complex mixtures with reduced sample pretreatment, short analysis times, relatively easy interpretation of results, and high sensitivity, fundamental in the study of paintings with very limited sample amounts.…”