Tyrannosaurus rex
Creators: name: Mike Buckley
name: Angela Walker
name: Simon Y. W. Ho
name: Yue Yang
name: Colin Smith
name: Peter Ashton
name: Jane Thomas Oates
name: Enrico Cappellini
name: Hannah Koon
name: Kirsty Penkman
name: Ben Elsworth
name: Dave Ashford
name: Caroline Solazzo
name: Phillip Andrews
name: John Strahler
name: Beth Shapiro
name: Peggy Ostrom
name: Hasand Gandhi
name: Webb Miller
name: Brian Raney
name: Maria Ines Zylber
name: M. Thomas P. Gilbert
name: Richard V. Prigodich
name: Michael Ryan
name: Kenneth F. Rijsdijk
name: Anwar Janoo
name: Matthew J. Collins
Publisher: name: Zenodo
Year: 2008
Subjects: Biodiversity
Taxonomy
Animalia
Chordata
Reptilia
Dinosauria
Tyrannosauridae
Tyrannosaurus
Tyrannosaurus rex

description: 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 gen­ erated 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 equiv­ alent 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 sensitiv­ ity 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 1/2) = ~ 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 T1/2 = 130 ky) (Fig. 1).   
 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 sufficient­ ly variable to enable meaningful comparison between distant taxa if enough sequence is ob­ tained (Fig. 2). Compared with ancient DNA amplification, contamination by collagen is in­ herently 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 ex­ cluded. 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 analy­ ses would have strengthened claims for the authenticity of the origin of the peptides (and potentially ameliorated the original problems of data interpretation) (4).   
 1BioArch, Departments of Biology, Archaeology, Chemistry and Technology Facility, University of York, Post Office Box 373, York YO10 5YW, UK. 2Department of Biological Chem­ istry, University of Michigan Medical School, Ann Arbor, MI 48109-0404, USA. ’ Evolutionary Biology Group, Department of Zoology, University of Oxford, OX1 3PS, UK. 4Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103, Leipzig, Germany. 5Department of Zoology, Michigan State University, East Lansing, MI 48824, USA. 6Department of Biology, Pennsylva- niaStateUniversity,UniversityPark, PA 16802,USA.7Center for Biomolecular Science and Engineering, University of California -Santa Cruz, CA 95064, USA. 8Department of Par­ asitology, Kuvin Center, Hebrew University of Jerusalem, Israel. 9BiologicalInstitute, UniversityofCopenhagen, Univer- sitetsparken 15, 2100 Copenhagen, Denmark. 10Chemistry Department, Trinity College, 300 Summit Street, Hartford, CT 06106,USA.11 ClevelandMuseumofNaturalHistory,1Wade Oval Drive, University Circle, Cleveland, OH 44106, USA. 12National Museum of Natural History “Naturalis,” P.O. Box 9517, 2300 RA Leiden, Netherlands. 13National Heritage Trust Fund Mauritius, Mauritius Institute, La Chausse'e Street Port Louis, Mauritius.   
*To whom correspondence should be addressed. E-mail: mc80@york.ac.uk  
 The remarkable soft-tissue preservation of the investigated T. rex specimen (MOR 1125) has been documented (8). However, microscop­ ic 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 experi­ ence, 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 assign­ ments (13) (table S1). A common diagenetic modification, deamidation, not considered in (2), may shed light on authenticity. The facile succinimide-mediated deamidation (14) of aspar­ agine occurred at N229G and N1156G in ostrich peptides (Ost 4 and Ost5) (see table S1 for nomenclature), presumably during sample prep­ aration. 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 a1(I) G362SEGPEGVR370, the deamidated (Q^F.!67) form of the sequence found in most mammals (12). By way of contrast, none of the three glutamine residues in the reported T. rex peptides are deamidated (table S1). Only time will tell if Q^E is a useful marker for authentically old collagen, but from the evidence presented, the  mastodon sequence looks more diagenetically altered than T. rex.    
 The unusual, fragmented nature of the re­ ported T. rex sequence does not make it ame­ nable to standard, model-based phylogenetic analysis. Instead, we examined the phylogenetic signal of the a1(I) frag­ ments of mastodon and T. rex using Neighbor- Net analysis and uncor­ rected genetic distances. Using the sequences reported in (13), both the T. rex and masto­ don signal display an affinity with amphibians (Fig. 2A). Our reinter­ pretation of the spectra (12) changes the affinity of mastodon but not of T. rex (Fig. 2B). In addition to the a1(I) peptides used in the Neighbor-Net analysis, Asara et al. reported two other peptides from T. rex (13); we question the interpretation of the a1(II) spectra (identical to frog) but not the a2(I) spectra (identical to chicken).   
 We require more data to be convinced of the authenticity of the T. rex collagen sequences re­ ported by Asara et al. Nevertheless, the hand­ ful of spectra reported for the temperate Pleisto­ cene mastodon fail nei­ ther phylogenetic nor diagenetic tests, thus highlighting the potential of protein mass spec­ trometry to bridge the present gulf in our un­ derstanding 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.
description: Published as part of Mike Buckley, Angela Walker, Simon Y. W. Ho, Yue Yang, Colin Smith, Peter Ashton, Jane Thomas Oates, Enrico Cappellini, Hannah Koon, Kirsty Penkman, Ben Elsworth, Dave Ashford, Caroline Solazzo, Phillip Andrews, John Strahler, Beth Shapiro, Peggy Ostrom, Hasand Gandhi, Webb Miller, Brian Raney, Maria Ines Zylber, M. Thomas P. Gilbert, Richard V. Prigodich, Michael Ryan, Kenneth F. Rijsdijk, Anwar Janoo & Matthew J. Collins, 2008, Comment on " Protein Sequences from Mastodon and Tyrannosaurus rex Revealed by Mass Spectrometry ", pp. 33 in Science 319 on page 33, DOI: 10.1126/science.1147046, http://zenodo.org/record/3746080