Ancient protein
Ancient proteins are complex mixtures and the term palaeoproteomics is used to characterise the study of proteomes in the past. Ancients proteins have been recovered from a wide range of archaeological materials, including bones, teeth, eggshells, leathers, parchments, ceramics, painting binders and well-preserved soft tissues like gut intestines. These preserved proteins have provided valuable information about taxonomic identification, evolution history, diet, health, disease, technology and social dynamics in the past.
Like modern proteomics, the study of ancient proteins has also been enabled by technological advances. Various analytical techniques, for example, amino acid profiling, racemisation dating, immunodetection, Edman sequencing, peptide mass fingerprinting, and tandem mass spectrometry have been used to analyse ancient proteins. The introduction of high-performance mass spectrometry in 2000 has revolutionised the field, since the entire preserved sequences of complex proteomes can be characterised.
Over the past decade, the study of ancient proteins has evolved into a well-established field in archaeological science. However, like the research of aDNA, it has been limited by several challenges such as the coverage of reference databases, identification, contamination and authentication. Researchers have been working on standardising sampling, extraction, data analysis and reporting for ancient proteins. Novel computational tools such as de novo sequencing and open research may also improve the identification of ancient proteomes.
History: the pioneers of ancient protein studies
Philip Abelson, Edgar Hare and Thomas Hoering
, Hare and Hoering were leading the studies of ancient proteins between the 1950s and the early 1970s. Abelson was directing the Geophysical Laboratory at the Carnegie Institute between 1953 and 1971, and he was the first to discover amino acids in fossils. Hare joined the team and specialised in amino acid racemisation. D/L ratios were used to date various ancient tissues such as bones, shells and marine sediments. Hoering was another prominent member, contributing to the advancement of isotopes and mass spectrometry. This golden trio drew many talented biologists, geologists, chemists and physicists to the field, including Marilyn Fogel, John Hedges and Noreen Tuross.Ralph Wyckoff
Wyckoff was a pioneer in X-ray crystallography and electron microscopy. Using microscopic images, he demonstrated the variability and damage of collagen fibres in ancient bones and shells. His research contributed to the understanding of protein diagenesis in the late 1960s, and highlighted that ancient amino acid profiles alone might not be sufficient for protein identification.Margaret Jope and Peter Wesbroek
and Wesbroek were leading experts in shell proteins and crystallisation. Wesbroek later established Geobiochemistry laboratory at the University of Leiden, focusing on biomineralisation and how this process facilitated protein survival. He also pioneered the use of antibodies for the study of ancient proteins in the 1970s and 1980s, utilising different immunological techniques such as Ouchterlony double immunodiffusion.Peggy Ostrom
Ostrom championed the use of mass spectrometry since the 1990s. She was the first to improve the sequence coverage of ancient proteins by combining different techniques such as peptide mass fingerprinting and liquid chromatography-tandem mass spectrometry.The biochemistry of ancient proteins
Formation & incorporation
Understanding how ancient proteins are formed and incorporated into archaeological materials are essential in sampling, evaluating contamination and planning analyses. Generally, for ancient proteins in proteinaceous tissues, notably, collagens in bones, keratins in wool, amelogenins in tooth enamel, and intracrystalline proteins in shells, they might be incorporated during the time of tissue formation. However, the formation of proteinaceous tissues is often complex, dynamic and affected by various factors such pH, metals, ion concentration, diet plus other biological, chemical and physical parameters. One of the most characterised phenomena is bone mineralisation, a process by which hydroxyapatite crystals are deposited within collagen fibres, forming a matrix. Despite extensive research, bone scaffolding is still a challenge, and the role of non-collagenous proteins remains poorly understood.Another category is complex and potentially mineralised tissues, such as ancient human dental calculi and ceramic vessels. Dental calculi are defined as calcified biofilms, created and mediated by interactions between calcium phosphate ions and a wide range of oral microbial, human, and food proteins during episodic biomineralisation. Similarly, the minerals of a ceramic matrix might interact with food proteins during food processing and cooking. This is best explained by calcite deposits adhering to the inside of archaeological ceramic vessels. These protein-rich mineralised deposits might be formed during repeated cooking using hard water and subsequent scaling.
Preservation
biomolecules like proteins are prone to degradation. For example, experimental studies demonstrate that robust, fibrous and hydrophobic keratins such as feathers and woollen fabrics decay quickly at room temperature. Indeed ancient proteins are exceptional, and they are often recovered from extreme burial contexts, especially dry and cold environments. This is because the lack of water and low temperature may slow down hydrolysis, microbial attack and enzymatic activities.There are also proteins whose chemical and physical properties may enable their preservation in the long term. The best example is Type 1 collagen; it is one of the most abundant proteins in skin and bone extracellular matrices. It is also mineralised, organised in a triple helix and stabilised by hydrogen bonding. Type 1 collagen has been routinely extracted from ancient bones, leathers, and parchments; these characteristics may contribute to its stability over time. Another common protein in the archaeological record is milk beta-lactoglobulin, often recovered from ancient dental calculi. Beta-lactoglobulin is a small whey protein with a molecular mass of around 18400 Da. It is resistant to heating and enzymatic degradation; structurally, it has a beta-barrel associated with binding to small hydrophobic molecules such as fatty acids, forming stable polymers.
Given that proteins vary in abundance, size, hydrophobicity, structure, conformation, function and stability, understanding protein preservation is challenging. While there are common determinants of protein survival, including thermal history, burial conditions and protein properties, there is no clear answer and protein diagenesis is still an active research field.
Structure & damage patterns
Generally, proteins have four levels of structural complexity: quaternary, tertiary, secondary and primary structure. Ancient proteins are expected to lose their structural integrity over time, due to denaturation or other diagenetic processes.Ancient proteins also tend to be fragmented, damaged and altered. Proteins can be cleaved into small fragments over time, since hydrolysis breaks peptide bonds. In terms of post-translational modifications, ancient proteins are often characterised by extensive damage such as oxidation, hydroxylation, deamidation, citrullination, phosphorylation, N-terminus glutamate to pyroglutamate and the addition of advanced glycation products to lysine or arginine. Among these modifications, glutamine deamidation is one of the most time-dependent processes. Glutamine deamidation is mostly a non-enzymatic process, by which glutamine is converted to glutamic acid via side-chain hydrolysis or the formation of a glutarimide ring. It is a slow conversion with a long half-time, depending on adjacent amino acids, secondary structures, 3D folding, pH, temperature and other factors. Bioinformatic tools are available to calculate bulk and site-specific deamidation rates of ancient proteins. The structural manifestation of these chemical changes within ancient proteins was first documented using scanning electron microscopy. Type-1 collagen protein fibrils of a permafrost-preserved woolly mammoth were directly imaged and shown to retain their characteristic banding pattern. These were compared against type-1 collagen fibrils from a temperate Columbian mammoth specimen. The Columbian mammoth collagen fibrils, unlike those of the permafrost-frozen woolly mammoth, had lost their banding, indicating substantial chemical degradation of the constituent peptide sequences. This also constitutes the first time that collagen banding, or the molecular structure for any ancient protein, has been directly imaged with scanning electron microscopy.
Palaeoproteomics
Overview
Palaeoproteomics is a fast-developing field that combines archaeology, biology, chemistry and heritage studies. Comparable to its high-profile sister field, aDNA analysis, the extraction, identification and authentication of ancient proteins are challenging, since both ancient DNA and proteins tend to be ultrashort, highly fragmented, extensively damaged and chemically modified.However, ancient proteins are still one of the most informative biomolecules. Proteins tend to degrade more slowly than DNA, especially biomineralised proteins. While ancient lipids can be used to differentiate between marine, plant and animal fats, ancient protein data is high-resolution with taxon- and tissue-specificities.
To date, ancient peptide sequences have been successfully extracted and securely characterised from various archaeological remains, including a 3.8 Ma ostrich eggshell, 1.77 Ma Homo erectus teeth, a 0.16 Ma Denisovan jawbone and several Neolithic pots. Hence, palaeoproteomics has provided valuable insight into past evolutionary relationships, extinct species and societies.