Abstract
Collagen is one of the most ubiquitous proteins in the animal kingdom and the dominant protein in extracellular tissues such as bone, skin and other connective tissues in which it acts primarily as a supporting scaffold. It has been widely investigated scientifically, not only as a biomedical material for regenerative medicine, but also for its role as a food source for both humans and livestock. Due to the long-term stability of collagen, as well as its abundance in bone, it has been proposed as a source of biomarkers for species identification not only for heat- and pressure-rendered animal feed but also in ancient archaeological and palaeontological specimens, typically carried out by peptide mass fingerprinting (PMF) as well as in-depth liquid chromatography (LC)-based tandem mass spectrometric methods. Through the analysis of the three most common domesticates species, cow, sheep, and pig, this research investigates the advantages of each approach over the other, investigating sites of sequence variation with known functional properties of the collagen molecule. Results indicate that the previously identified species biomarkers through PMF analysis are not among the most variable type 1 collagen peptides present in these tissues, the latter of which can be detected by LC-based methods. However, it is clear that the highly repetitive sequence motif of collagen throughout the molecule, combined with the variability of the sites and relative abundance levels of hydroxylation, can result in high scoring false positive peptide matches using these LC-based methods. Additionally, the greater alpha 2(I) chain sequence variation, in comparison to the alpha 1(I) chain, did not appear to be specific to any particular functional properties, implying that intra-chain functional constraints on sequence variation are not as great as inter-chain constraints. However, although some of the most variable peptides were only observed in LC-based methods, until the range of publicly available collagen sequences improves, the simplicity of the PMF approach and suitable range of peptide sequence variation observed makes it the ideal method for initial taxonomic identification prior to further analysis by LC-based methods only when required.
Highlights
Collagen has been considered an important biomolecule with biomedical uses, such as the base of a scaffold for tissue regeneration [1,2], as well as in the food industry in its denatured form of gelatine [3,4]
As the most abundant protein in the extracellular tissues used in animal feed [5], it was widely used as a cheap source of protein for livestock feed made from a range of animal species that included livestock but occasionally fallen exotic animals from zoological gardens and country parks
Following the outbreak of variant Creutzfeldt–Jakob disease, believed caused by the consumption of food contaminated with prions such as cattle tissues of individuals that suffered from bovine spongiform encephalopathy, and the subsequent change in the regulations over the use of processed animal by-products during the 1990s [6], the ability to discriminate the species of the tissues going into the feed became a global concern [7,8]
Summary
Collagen has been considered an important biomolecule with biomedical uses, such as the base of a scaffold for tissue regeneration [1,2], as well as in the food industry in its denatured form of gelatine [3,4]. Following the outbreak of variant Creutzfeldt–Jakob disease, believed caused by the consumption of food contaminated with prions such as cattle tissues of individuals that suffered from bovine spongiform encephalopathy, and the subsequent change in the regulations over the use of processed animal by-products during the 1990s [6], the ability to discriminate the species of the tissues going into the feed became a global concern [7,8] It is the most abundant protein to survive into the archaeological and palaeontological records, which can be informative of early forms of animal husbandry [9] as well as environmental changes that occurred during the past [10]. Collagen survival is thought to be enhanced due to the confinement of the triple helix within its mineral bioapatite [16]
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