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Watching forensic scientists perform on TV series such as Bones and CSI gives the impression that there is always a solution to any problem put before them, which is generally reached via a tense, exciting process. That is one reason why there is a glut of new and recent forensic graduates out there vying for jobs in a highly competitive market. Of course, the forensic process is not quite as easy as portrayed and all samples do not have that distinctive feature that solves the case. Take bone, for example. In reality, there might well be just a fragment of bone to examine, insufficient to tell its own story and so small that even the species it originated from cannot be identified. This identity problem is not confined to the forensic world. Recognising the bone species is also vitally important in other fields of work such as food safety, trading standards, archaeology and palaeontology. Scientists from the University of York, UK, have taken a chemical approach to species identification, using the protein collagen, which is found in bone connective tissue. Collagen has extraordinary stability. Recent studies have revealed that it can survive being heated to 145°C during rendering of meat and bone meal (MBM). More remarkably, perhaps, collagen has been extracted and sequenced from dinosaur bones more than 80 million years old. This stability suggests that most bone samples brought in for inspection should have collagen available for examination and the York scientists, Michael Buckley, Matthew Collins, Jane Thomas-Oates and Julie Wilson, took advantage of this. They assembled bone specimens from 32 different mammalian species, including domestic and farm animals, wild animals and some archaeological samples. In addition, they analysed rendered cattle, pig, sheep and chicken MBM. Bones from four birds, chicken, turkey, duck and pheasant, were also analysed as the biological class most closely related to mammals. Collagen was extracted from powdered bone samples following dimineralisation and gelatinisation. The protein was digested with trypsin to produce a series of peptides which were the main target for analysis. The expectation was that the structure of collagen would vary from species to species, so that at least some of the tryptic peptides would be species-specific. Some reported studies on collagen extracted from bones have used LC/MS to separate and analyse the peptides but the researchers chose what they regarded as a simpler route. They used solid-phase extraction with C18 ZipTips, eluting two peptide fractions with 10% and 50% acetonitrile in aqueous trifluoroacetic acid. The two fractions acquired from each bone sample were analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) over the extended range of m/z 800-3200. The collagen peptide m/z values were noted and compared by cluster analysis based on the presence or absence of m/z values. The sequences of selected peptides were also determined by tandem mass spectrometry. In total, 92 peptides were identified as markers for species identification. For the birds, 12 m/z values found in samples could be markers for avian samples. Within the avian specimens, 3 m/z values were found only for chicken, pheasant and turkey and four were found only for chicken and pheasant. This indicates the potential of the peptide marker method. The cluster analysis revealed that 26 out of the 32 mammals could be identified from the peptides. In the other cases, the level of family was ascertained. The genus failures were hippopotamus/pygmy hippopotamus, fallow deer/red deer/elk, and African/Indian elephant. The researchers declared that the analysis of more replicates might identify more markers that would extend the method to these species. All identifications would be improved by complementary sequence information for the peptides to confirm that a specific peptide was homologous. This process would also lead to the identification of new biomarkers for certain species, which occurred in several cases in this study. The data for modern sheep, goat, deer and elk specimens were used as a training set in the multivariate analysis of archaeological samples sourced from ancient pottery. Those identified as sheep and goat by morphological means were correctly classified from their collagen peptide m/z values and another was classified as the deer family (Cervidae). Some of the specimens had been buried for about 11,000 years in a warm climate, giving a thermal age of more than 100 kacollagen@10°C. This parameter is a factor of burial depth and temperature, as well as age, and its significance here lies in the fact that DNA survival appears to be possible only at thermal ages less than 50 kaDNA@10°C. So, collagen analysis can succeed where DNA analysis fails. It also removes the contamination risk encountered during DNA amplification. Collagen also survived heating to 145°C during the MBM rendering of animal carcasses to produce animal feed. Chicken MBM was identified as avian, cattle MBM as bovine and pig MBM as porcine. The results for sheep MBM were not so precise since two key, unique peptides were not observed in the mass spectra. Nevertheless, they were classified as sheep or goat, which is still useful, since the distinction between ruminants and non-ruminants is a legal requirement for MBM. This technique has broad application to any area where species identity is important, such as food authenticity, food safety, forensics and archaeology. It could also be extended to other species such as fish, birds and amphibians, as long as unique peptides were produced from their collagen during digestion with trypsin.
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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Collagen was even extracted from dinosaur bones |
