Creation of a peptide database of corneous beta-proteins of marine turtles for the identification of tortoiseshell: archaeological combs as case study

Tortoiseshell is a proteinaceous material derived from the scutes of marine turtles, and was shaped into an abundance of objects, especially luxurious items, at its peak in the seventeenth and eighteenth century. It has continued to be used even after the advent of plastics and remains one of the main causes of illegal poaching of marine turtles, in particular the hawksbill turtle Eretmochelys imbricata. Tortoiseshell is made of structural proteins, of which the most abundant are known as β-keratins, or ‘corneous beta-proteins' (CBPs), a family of short proteins containing a central structure in β-sheets. There are, however, few CBP sequences of marine turtles in protein databases. The scutes of the five main species of marine turtles (Chelonia mydas, Caretta caretta, Eretmochelys imbricata, Lepidochelys olivacea and Lepidochelys kempii) were analysed by proteomics, using nano-liquid chromatography-Orbitrap-mass spectrometry to generate peptidic markers for species identification. A total of 187 marker sequences were identified, the large majority of them obtained from automated de novo sequencing. The sequences were classified into peptides A to F: A to D at the N-terminus and central region that forms the β-pleated sheets, E1–4 for a variable region of glycine-repeats region and F at the C-terminus. The markers were tested against a set of combs discovered in various archaeological sites of modern period in France, successfully identifying hawksbill turtle and highlighting patterns of degradation in archaeological tortoiseshell.