The Demodifier: A tool for screening modification-induced alternate peptide taxonomy in palaeoproteomics
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Abstract
In palaeoproteomic research, the accuracy of taxonomic matches is crucial, as research questions frequently hinge on which species were utilised by ancient people. However, protein modifications including deamidation of glutamine and asparagine, and conversion of glutamine or glutamic acid to pyroglutamic acid, can change the sequence of peptides, leading to spurious taxonomic detections and potentially inaccurate archaeological interpretations. While a handful of examples of this phenomenon have been reported in the literature, the issue is potentially much wider reaching than currently realised. In reality, any time a peptide containing a deamidated glutamine or asparagine residue, an unmodified glutamic acid or aspartic acid residue, or a pyroglutamic acid modification is detected by proteomic search software, the sequence, and therefore potentially its taxonomy, may be incorrect, which could potentially lead to unsound archaeological interpretations. The Demodifier is a fast, open source tool which solves this issue by screening for modification-induced alternate peptide taxonomy, enabling archaeologists to make informed interpretations of the taxonomies of peptides detected in ancient samples. To assess its utility, The Demodifier is tested against an archaeological dataset containing all unique peptides reported in palaeoproteomic studies of dental calculus and vessels. The results reveal that modification-induced alternate peptide taxonomies are severely under-reported, occurring almost ten times more frequently than previously understood. Modifications were found to produce three different types of inaccurate taxonomic matches: those which yielded completely different taxonomic lowest common ancestors to the input peptide, those which were more taxonomically specific than the input peptide, and those which were less taxonomically specific than the input peptide. The Demodifier therefore enables the rapid detection of potentially inaccurate peptide taxonomies, avoiding spurious archaeological interpretations in future studies.
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Over the last decades, mass spectrometry-based proteomics has been widely adopted in archaeology as a mean for taxonomic identification. However, the accurate detection of peptides in ancient protein studies can be challenging. The occurrence of degradation patterns and post-translational modifications (PTMs) in peptide sequences, notably deamidation of glutamine (Q) to glutamic acid (E) and asparagine (N) to aspartic acid (D), or the conversion of glutamine or glutamic acid to pyroglutamic acid can indeed blur identifications. In some instances, these substitutions can potentially lead to incorrect taxonomic assignments when an amino acid is transformed into another with identical mass, making different variants of a peptide indistinguishable. A well-known case in palaeoproteomics is the frequently detected whey β-lactoglobulin …
Over the last decades, mass spectrometry-based proteomics has been widely adopted in archaeology as a mean for taxonomic identification. However, the accurate detection of peptides in ancient protein studies can be challenging. The occurrence of degradation patterns and post-translational modifications (PTMs) in peptide sequences, notably deamidation of glutamine (Q) to glutamic acid (E) and asparagine (N) to aspartic acid (D), or the conversion of glutamine or glutamic acid to pyroglutamic acid can indeed blur identifications. In some instances, these substitutions can potentially lead to incorrect taxonomic assignments when an amino acid is transformed into another with identical mass, making different variants of a peptide indistinguishable. A well-known case in palaeoproteomics is the frequently detected whey β-lactoglobulin peptide TPEDD/NEALEK, where the Bovinae peptide with unmodified D is indistinguishable from the deamidated N Ovis version (Warinner et al. 2014), which can influence our understanding of ancient dairy exploitation, diet and culinary practices.
Other Modification-Induced Sequence Permutations (MISPs) have been reported in the literature but systematic screening for MISPs and their taxonomies is not yet standard practice and has so far been conducted manually. To address this issue, Evans (2025) presents a new tool, The Demodifier, which screens for all potential MISPs of a given set of peptides and then provides alternative taxonomies for each one. The author tests this tool on a large, published dataset of dietary peptides found in archaeological dental calculus and ceramic vessels. The results show that MISPs appear to be 15% more common than previously reported. In some of these cases, this resulted in completely different taxonomic detections or more or less specific identifications.
Thus, The Demodifier is a valuable tool for verifying taxonomic assignments and avoiding inaccurate archaeological interpretations of proteomic results. In addition, it brings into focus an issue that is likely to be much more widespread than previously realised in palaeoproteomics studies and that warrants further research and discussion. However, as Evans acknowledges, The Demodifier is not designed to replace standard steps in a palaeoproteomics pipeline, but rather to be used as an additional taxonomic validation tool, and some of the current challenges could be addressed in future versions.
Entirely open-source, Evans has created an incredibly valuable tool for the wide palaeoproteomics community, that she accompanies with a most useful tutorial. In short, The Demodifier is surely going to become a reference tool in proteomics for archaeology.
References
Evans, M.A. (2025) The Demodifier: a tool for screening modification-induced alternate peptide taxonomy in palaeoproteomics. bioRxiv, ver.4 peer-reviewed and recommended by PCI Archaeology https://doi.org/10.1101/2025.01.09.632126
Warinner, C., Hendy, J., Speller, C., Cappellini, E., Fischer, R., Trachsel, C., Arneborg, J., Lynnerup, N., Craig, O.E., Swallow, D.M., Fotakis, A., Christensen, R.J., Olsen, J.V., Liebert, A., Montalva, N., Fiddyment, S., Charlton, S., Mackie, M., Canci, A., Bouwman, A., Rühli, F., Gilbert, M.T.P. and Collins, M.J. (2014). Direct evidence of milk consumption from ancient human dental calculus. Scientific Reports 4, 1–6. https://doi.org/10.1038/srep07104
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