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Technical CommentsComment on "Protein Sequences from Mastodon and Tyrannosaurus rex Revealed by Mass Spectrometry"
We used authentication tests developed for ancient DNA to evaluate claims by Asara et al. (Reports, 13 April 2007, p. 280) of collagen peptide sequences recovered from mastodon and Tyrannosaurus rex fossils. Although the mastodon samples pass these tests, absence of amino acid composition data, lack of evidence for peptide deamidation, and association of
1 BioArch, Departments of Biology, Archaeology, Chemistry and Technology Facility, University of York, Post Office Box 373, York YO10 5YW, UK. 1(I) collagen sequences with amphibians rather than birds suggest that T. rex does not.
2 Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109–0404, USA. 3 Evolutionary Biology Group, Department of Zoology, University of Oxford, OX1 3PS, UK. 4 Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103, Leipzig, Germany. 5 Department of Zoology, Michigan State University, East Lansing, MI 48824, USA. 6 Department of Biology, Pennsylvania State University, University Park, PA 16802, USA. 7 Center for Biomolecular Science and Engineering, University of California–Santa Cruz, CA 95064, USA. 8 Department of Parasitology, Kuvin Center, Hebrew University of Jerusalem, Israel. 9 Biological Institute, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark. 10 Chemistry Department, Trinity College, 300 Summit Street, Hartford, CT 06106, USA. 11 Cleveland Museum of Natural History, 1 Wade Oval Drive, University Circle, Cleveland, OH 44106, USA. 12 National Museum of Natural History "Naturalis," P.O. Box 9517, 2300 RA Leiden, Netherlands. 13 National Heritage Trust Fund Mauritius, Mauritius Institute, La Chaussée Street Port Louis, Mauritius. * To whom correspondence should be addressed. E-mail: mc80{at}york.ac.uk Early reports of DNA preservation in multimillion-year-old bones (i.e., from dinosaurs) have been largely dismissed (1, 2) (table S1), but reports of protein recovery are persistent [see (3) for review]. Most of these studies used secondary methods of detection, but Asara et al.(2) recently reported the direct identification of protein sequences, arguably the gold standard for molecular palaeontology, from fossil bones of an extinct mastodon and Tyrannosaurus rex. After initial optimism generated by reports of dinosaur DNA, there has been increasing awareness of the problems and pitfalls that bedevil analysis of ancient samples (1), leading to a series of recommendations for future analysis (1, 4). As yet, there are no equivalent standards for fossil protein, so here we apply the recommended tests for DNA (4) to the authentication of the reported mastodon and T. rex protein sequences (2)(Table 1).
First, the likelihood of collagen survival needs to be considered. The extremely hierarchical structure of collagen results in unusual, catastrophic degradation (5) as a consequence of fibril collapse. The rate of collagen degradation in bone is slow because the mineral "locks" the components of the matrix together, preventing helical expansion, which is a prerequisite of fibril collapse (6). The packing that stabilizes collagen fibrils (6) also increases the temperature sensitivity of degradation (Ea 173 kJ mol–1) (Fig. 1). Collagen decomposition would be much faster in the T. rex buried in the then-megathermal (>20°C) (7) environment of the Hell Creek formation [collagen half-life (T
This risk of contamination also needs to be evaluated. Collagen is an ideal molecular target for this assessment because the protein has a highly characteristic motif that is also sufficiently variable to enable meaningful comparison between distant taxa if enough sequence is obtained (Fig. 2). Compared with ancient DNA amplification, contamination by collagen is inherently less likely. Furthermore, because the bones sampled in (2) were excavated by the authors, obvious contamination sources such as animal glue (used in conservation) can be excluded. However, concentrating protein from the large amounts of bone used (2.5 g) may have heightened the risk of extraneous proteins entering the sample during extraction, although there have been no systematic studies of this phenomenon. Independent extraction and analyses would have strengthened claims for the authenticity of the origin of the peptides (and potentially ameliorated the original problems of data interpretation) (4).
The remarkable soft-tissue preservation of the investigated T. rex specimen (MOR 1125) has been documented (8). However, microscopic preservation does not equate with molecular preservation (9). Immunohistochemistry provides support for collagen preservation, but Asara et al. (2) presented no data regarding inhibition assays with collagen from different species or cross-reactivity with likely contaminants [e.g., fungi (10)]. Curiously, no amino acid compositional analysis was conducted [see (11)], although immonium ions were identified by time-of-flight secondary ion mass spectrometry. In our experience, collagen-like amino acid profiles have been obtained in all bones from which we could obtain collagen sequence (Fig. 1, inset).
Regarding the proof of sequence authenticity, the spectra reported by Asara et al. (12) are inconsistent with some of the sequence assignments (13) (table S1). A common diagenetic modification, deamidation, not considered in (2), may shed light on authenticity. The facile succinimide-mediated deamidation (14) of asparagine occurred at N229G and N1156G in ostrich peptides (Ost 4 and Ost5) (see table S1 for nomenclature), presumably during sample preparation. Direct hydrolytic deamidation is slower (14), and an expectation of elevated levels of such products is reasonable for old samples. We agree with the most recent interpretation (13) of the spectrum illustrated in Fig. 2B as
The unusual, fragmented nature of the reported T. rex sequence does not make it amenable to standard, model-based phylogenetic analysis. Instead, we examined the phylogenetic signal of the We require more data to be convinced of the authenticity of the T. rex collagen sequences reported by Asara et al. Nevertheless, the handful of spectra reported for the temperate Pleistocene mastodon fail neither phylogenetic nor diagenetic tests, thus highlighting the potential of protein mass spectrometry to bridge the present gulf in our understanding between the fate of archaeological and fossil proteins. To avoid past mistakes of ancient DNA research (1), we recommend that future fossil protein claims be considered in light of tests for authenticity such as those presented here.
Supporting Online Materialwww.sciencemag.org/cgi/content/full/319/5859/33c/DC1 SOM Text Table S1 References
Received for publication 26 June 2007. Accepted for publication 20 November 2007.
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2 thousand years (ky] than it would have been in the mastodon lying within the Doeden Gravel Beds (present-day mean temperature, 7.5°C; collagen T
E367) form of the sequence found in most mammals (Science. ISSN 0036-8075 (print), 1095-9203 (online)
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