Exploring patterns of diagenesis in ancient proteins

Palaeoproteomics is a rapidly growing field of research that involves applying mass spectrometry to objects of archaeological and palaeontological interest in order to gain insight into the past. However, due to a lack of clear authentication criteria and understanding of the biomolecular characteristics of ancient proteins, there are suspicions about many remarkable results. This is of particular importance, as many samples are derived from museum collections, which may have been exposed to contamination, or had contaminants added as part of past conservation treatments. This thesis aims to (a) investigate the diagenetic forces acting on ancient proteins by quantifying the patterns of degradation in truly ancient proteins, and (b) use this knowledge in order to develop tools to authenticate results. This thesis develops a novel tool, deamiDATE 1.0, for the authentication of ancient proteins using site-specific deamidation. This is relatively effective in broadly differentiating between modern and ancient proteins (with a focus on structural (collagen) and dietary (milk) proteins). Analysis of site-specific deamidation in ancient milk proteins reveals that although there is a modest trend of increasing deamidation over time, the deamidation of milk proteomes in the archaeological record is hugely variable. Therefore, other lines of evidence are recommended in order to assess the authenticity of low abundance proteins. Lastly, the patterns of collagen degradation in parchment are elucidated, showing that there is a region of the collagen structure that is a hotspot for hydrolysis. Taken together, the results of this thesis represent a novel contribution to the understanding of the diagenetic forces that act on proteins through time, and how to reliably authenticate proteomics data

Gene network analyses support subfunctionalization hypothesis for duplicated hsp70 genes in the Antarctic clam

Funder: Natural Environment Research Council; doi: http://dx.doi.org/10.13039/501100000270Funder: Wolfson College, University of Cambridge (GB)Abstract: A computationally predicted gene regulatory network (GRN), generated from mantle-specific gene expression profiles in the Antarctic clam Laternula elliptica, was interrogated to test the regulation and interaction of duplicated inducible hsp70 paralogues. hsp70A and hsp70B were identified in the GRN with each paralogue falling into unique submodules that were linked together by a single shared second neighbour. Annotations associated with the clusters in each submodule suggested that hsp70A primarily shares regulatory relationships with genes encoding ribosomal proteins, where it may have a role in protecting the ribosome under stress. hsp70B, on the other hand, interacted with a suite of genes involved in signalling pathways, including four transcription factors, cellular response to stress and the cytoskeleton. Given the contrasting submodules and associated annotations of the two hsp70 paralogues, the GRN analysis suggests that each gene is carrying out additional separate functions, as well as being involved in the traditional chaperone heat stress response, and therefore supports the hypothesis that subfunctionalization has occurred after gene duplication. The GRN was specifically produced from experiments investigating biomineralization; however, this study shows the utility of such data for investigating multiple questions concerning gene duplications, interactions and putative functions in a non-model species

Permafrost preservation reveals proteomic evidence for yak milk consumption in the 13th century

Domesticated yaks endure as iconic symbols of high-altitude frozen landscapes, where herding communities depend on their high-fat milk, transport, dung, and natural fibers. While there is established proteomic evidence for ancient consumption of ruminant and horse milk in the mountains and steppes of northern Eurasia, yak dairy products have yet to be detected. Yak domestication and the species’ dispersal from Tibet into the mountainous zones to the north are also poorly resolved due to a paucity of zooarchaeological data. To examine the potential of paleoproteomics to shed light on domesticated yak in Mongolia, we analyzed human dental calculus from Mongol era elite individuals recovered from permafrost burials in Khovsgol province, where people continue to herd yak to this day. We report the first evidence for yak dairy consumption, linked to local resource control. In addition, we confirm a large diversity of recovered whey, curd, tissue, and blood proteins, likely reflecting the excellent preservation conditions found at permafrost sites

Assessing the degradation of ancient milk proteins through site-specific deamidation patterns.

The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG  recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data

New insights into Neolithic milk consumption through proteomic analysis of dental calculus

There has long been debate over the origins of dairy consumption within European populations. Whilst it was previously assumed that lactase persistence (LP) was under positive selection following the advent of agriculture, recent genetic studies of prehistoric human remains have revealed LP may have only emerged in Europe in the last 4000 years. These findings stand in contrast to organic residue analysis of Neolithic pottery indicating the utilisation of dairy products, and zooarchaeological mortality profiles consistent with dairying herds at Neolithic sites. The recent discovery of the milk protein β-lactoglobulin (BLG) within human dental calculus presents a new method via which to explore dairy product consumption in the archaeological past. Here, we apply shotgun proteomic analysis to dental calculus samples from three British Neolithic sites, revealing the earliest identification of BLG in human dental calculus to date. The presence of BLG peptides in individuals who are unlikely to possess LP provides new insight into dairying in the British Neolithic, suggesting the potential processing of milk by Neolithic populations to reduce the lactose content of dairy products

Assessing the degradation of ancient milk proteins through site-specific deamidation patterns.

The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG  recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data