Enamel peptides reveal the sex of the Late Antique ‘Lovers of Modena’

Abstract

Recent work has disclosed the critical role played by enamel peptides in sex classification of old skeletal remains. In particular, protein AMELY (amelogenin isoform Y) is present in the enamel dental tissue of male individuals only, while AMELX (isoform X) can be found in both sexes. AMELY can be easily detected by LC-MS/MS in the ion extracted chromatograms of the SM(ox)IRPPY peptide (monoisotopic [M + 2 H]+2 mass = 440.2233 m/z). In this paper, we exploited the dimorphic features of the amelogenin protein to determine the sex of the so-called ‘Lovers of Modena’, two Late Antique individuals whose skeletons were intentionally buried hand-in-hand. Upon discovery, mass media had immediately assumed they were a male-female couple, even if bad preservation of the bones did not allow an effective sex classification. We were able to extract proteins from the dental enamel of both individuals (~1600 years old) and to confidently classify them as males. Results were compared to 14 modern and archaeological control samples, confirming the reliability of the ion chromatogram method for sex determination. Although we currently have no information on the actual relationship between the ‘Lovers of Modena’ (affective? Kin-based?), the discovery of two adult males intentionally buried hand-in-hand may have profound implications for our understanding of funerary practices in Late Antique Italy.

A comparison of proteomic, genomic, and osteological methods of archaeological sex estimation

“…Following removal of these non-specific amelogenin peptides, signals of all peptides unambiguously attributed to either AMELX or AMELY are then combined into a single measure 33 . This process differs from the methods of Stewart et al 34,36,57 , Wasinger et al 35 , or Froment et al 54 , which relied on detection of two or four unique peptide masses only. In contrast, the proteomic method employed here identifies and sums signal intensities of multiple different AMELX and AMELY peptides with various permutations of common post translational modifications (PTMs), such as deamidation or oxidation.…”

Palaeoproteomics gives new insight into early southern African pastoralism

“…In addition to ancient DNA studies, the past few years have witnessed a multiplicity of analyses based on the extraction and characterisation of long-term persistent biomolecules: proteins [56][57][58][59][60][61] . Since their amino acid sequence is coded for by the DNA, proteins carry genetically derived features that allow researchers to propose phylogenetic relationships of extinct species [62][63][64][65] , sex attribution 66,67 or even characterisation of palaeopathogens 68 . The major protein of bone and teeth, type I collagen (constituting 90% of the organic phase of these materials), is composed of amino acid sequences that carry taxon-related differences in their order, allowing species discrimination 69 .…”

Tooth Enamel and Its Dynamic Protein Matrix

“…The power of enamel proteins and proteomics as a robust method for sex identification is demonstrated in the case of the “Lovers of Modena,” in which the determination of sex allows us to see history under a different perspective [ 43 ]. In the cemetery of Modena (Italy), the burial site of two adults holding hands was uncovered and dated to the 4th-6th century.…”

“…Another hurdle is that there might be tradeoffs between precision in sampling methods and accuracy in protein isolation that are of key importance to avoid loss of proteins, contaminations, introduction of false positives and compromised integrity of proteins and data. It is encouraging though, that proteome analyses of human fossil material demonstrate the feasibility to obtain data on protein composition in fossil human enamel [ 43 , 44 , 46 , 54 ].…”

“…Schematic of treatment steps used to characterize proteins in human tooth enamel from archeological, modern and teeth affected by molar hypomineralization (chalky tooth enamel). Porto et al [ 21 ]; Lugli et al [ 43 ]; Stewart et al [ 44 ]; Castiblanco et al [ 45 ]; Stewart et al [ 46 ]; Jagr et al [ 47 ]; Farah et al [ 48 ]; Nielsen-Marsh et al [ 49 ]; Mangum et al [ 50 ]. Each bracket denotes alternative options and reflects the variability in published protocols.…”