Investigating protein survival into deep time and the potential of proteomics in palaeontology

Palaeoproteomics is a relatively new field of research in archaeological science, but has great potential for phylogenetic analysis of fossil taxa, particularly beyond the preservation limit of ancient DNA. This PhD collects proteomic data from a wide variety of fossils (e.g. bone, eggshell, soft tissue), from a range of localities (e.g. Pleistocene tar seeps, Antarctica) and ages (Holocene-Eocene). The resulting data is used to evaluate different proteomic methods for the successful retrieval of palaeo-proteins, and what these proteins can inform us about protein preservation mechanisms and use in phylogenetic reconstructions. Testing protein endogeneity is also considered, as this is a key area of research in palaeoproteomics. Different methods of screening samples for further proteomic analysis was assessed, and the use of enzymes other than trypsin, as well as gel-based extraction methods was investigated to maximise protein recovery. Protein survival is dependent on a number of factors, including environment, age and the substrate in which the proteomic information is preserved. The age of the fossil was found to be more of an influencing factor than the environment. A number of considerations are presented for investigating the endogeneity of fossil peptides. The data obtained from fossil collagen sequences were successfully used to reconstruct the relationships between extinct sloths and their extant relatives (Choloepus sp. and Bradypus sp.), with the results suggesting a new placement of both extant taxa in relation to their ancestors. The use of non-collagenous proteins in phylogenetic reconstruction was also considered, but low sequence coverage prevents successful phylogenetic resolution. This thesis provides recommendations for sample screening and testing peptide endogeneity as well as showcasing the use of proteomics in palaeontology.