Understanding protein diagenesis in tooth enamel for palaeoproteomic potential

Tooth enamel palaeoproteomics is an emerging field in palaeontology and archaeology. Understanding the diagenesis of the enamel palaeoproteome is fundamental to develop optimal proteome extraction methods for phylogenetic analysis and sex estimation. Combining chiral amino acid analysis with soft ionisation protein mass spectrometry provides deeper insights into fossil protein. In this thesis, these two analytical approaches are used to test optimal protocols for enamel palaeoproteomics and applied to highly degraded samples (burnt and of Pleistocene & Miocene age). Testing of different pre-treatment methods (bleached vs unbleached) and preparation protocols (digestion free vs trypsin digestion) showed that the best approach for enamel preparation for maximum proteomic information recovery was the unbleached, digestion free approach. Trypsin digested enamel resulted in poorer recoveries of fossil enamel proteome. For modern enamel, if ameloblastin is of particular interest, then bleaching is recommended.
The heating effect on enamel proteins was investigated on experimentally heated samples. Protein diagenesis patterns for modern and fossil enamel were characterised and suggested selection criteria for likely successful peptide matching in four taxa (mammoth, rhino, horse & cow) were determined. When tested on naturally heated/cremated enamel, three out of 20 heated samples still contained the relevant peptides for sex estimation. Three fossil enamel case studies: 20+ Ma Rhinoceratidae, 8 Ma Oreopithecus & Maremmia, and two important South African hominins (Homo naledi and Australopithecus africanus) were investigated for their intra-crystalline protein diagenesis by chiral amino acid analysis. The chiral amino acid analysis results aligned with proteomic data obtained on the same samples in collaboration. This establishes the importance of chiral amino acid analysis as a reliable authenticating approach for assessing the presence of endogenous amino acids and peptides in fossil enamel. This thesis therefore demonstrates the value of combining multiple analytical techniques to improve the understanding of enamel palaeoproteome for phylogeny and sex-estimation.