Expansion for the <i>Brachylophosaurus canadensis</i> Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein

Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a Brachylophosaurus canadensis specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, B. canadensis is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, B. canadensis is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of B. canadensis collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the previous study with substantially less sample material. Data are available via ProteomeXchange with identifier PXD005087.序列数据源自DNA与蛋白质等生物分子，可为进化研究提供关键信息，此前一直被认为完全无法应用于恐龙古生物学研究。相较于DNA，蛋白质被预测具有更长的存续寿命，目前已有从两例非鸟类恐龙中获取蛋白质的报道，但这些结果仍存在争议。若要使源自已灭绝中生代生物的蛋白质组学（proteomic）数据在系统发育与古生物生物学问题研究中发挥最大潜力，必须证明可从化石中可靠且可重复地获取肽序列，且古蛋白质的片段序列可得到持续扩展。为验证"可从数百万年历史的化石组织中反复检测并验证肽序列"这一假说，我们对2009年曾从中获取I型胶原肽的短冠龙（Brachylophosaurus canadensis）标本（MOR 2598）应用了更新后的提取方法、高分辨率质谱（high-resolution mass spectrometry）分析与生物信息学（bioinformatics）分析。本次研究共获取8条I型胶原肽序列，其中2条与2009年获取的肽序列一致，另有6条为全新肽序列。系统发育分析将获取的序列归类于基干主龙类（basal archosauria）。仅考虑新序列时，短冠龙与鳄形类（crocodylians）亲缘关系更近；而当分析所有序列（本研究当前序列及2009年报道的序列）时，短冠龙则与基干鸟类亲缘关系更近。本数据有力支持了"这些肽序列源自内源"的假说，证实肽可在数千万年历史的标本中存续，并强化了2009年研究的可信度。此外，新数据扩展了短冠龙I型胶原的序列覆盖度：I型胶原α1链的覆盖度提升33.6%，α2链提升116.7%。最后，本研究证实了采用更新方法与仪器设备重新审视此前研究过的标本的重要性——我们仅使用极少量样品，就获得了与此前研究相当的序列数据。相关数据可通过ProteomeXchange获取，标识符为PXD005087。