SGER: A Method to Sequence Novel Peptides from Unsequenced Taxa: Soft tissue of the 68M Year Old Tyrannosaurus rex

这笔小额的探索性研究经费将允许对一种新颖的、基于生物信息学和质谱的从头肽测序方法进行初步研究，以对蛋白质或基因组内容未被测序的分类群中的保守蛋白质进行测序。该方法将提供分子系统发育假说，并有可能证明古代化石中的肽含量。这些信息将有助于阐明未被鉴定的分类群的系统发育，并将为生物学和古生物学研究者提供有用的信息。该策略依赖于对肽串联质谱的数据库搜索，而不是基于邻近分类群中给定蛋白质的已知序列假设生成的预测蛋白质序列。数百个预测的蛋白质序列数据库条目将使用基于生物信息学的方法生成，如序列比对、简单加权共识和点辅助突变矩阵，并自动用于网络上的共享软件。使用基于质谱的蛋白质组学（如Sequest）的数据库评分算法，通过串联质谱与预测蛋白质序列的明确匹配，将发现新的肽序列。新序列，独特的问题分类单元，将严格验证通过分数截止和人工检查，然后通过合成所得的推定肽串联质谱分析和RNA测序的测试分类单元，如果可能的话。该分析将用于包括鸵鸟和短吻鳄在内的几个现代未测序分类群。该方法最终将用于对2003年在蒙大拿州发现的6800万年前的霸王龙化石软组织中的独特肽进行测序。这项工作不会（事实上，也不可能）得出霸王龙的完整基因序列——我们没有一整套霸王龙的蛋白质（蛋白质组），即使肽存活下来，它们也会被转录和翻译水平从实际的DNA代码中分离出来，而这些转录和翻译水平确实会随着时间的推移而消失。这种研究化石的新方法的优点是，非常低水平的蛋白质物质，包括修饰的蛋白质，可以用高灵敏度和快速扫描的质谱仪以高通量的方式进行测序。一旦建立了许多独特蛋白质序列的数据库，该方法就可以作为一种准确识别化石生物的手段。它还将帮助研究那些基因组内容尚未测序的未灭绝生物的研究人员，并将为关于肽和蛋白质分子钟的假设提供具体的测试。此外，对化石材料的研究将打开一扇通往分子水平解剖学的新窗口，并为恐龙分子系统发育的研究开创先例。

This small grant for exploratory research will allow preliminary work on a novel, bioinformatics and mass spectrometry based de novo peptide sequencing method to sequence conserved proteins from taxa whose protein or genomic content has not been previously sequenced. The method will provide molecular phylogenetic hypotheses and potentially demonstrate peptide content in ancient fossils. This information will help elucidate the phylogeny of uncharacterized taxa, and will provide useful information for biological and paleontological researchers. The strategy relies upon database searching of tandem mass spectra of peptides versus hypothetically generated predicted protein sequences based on known sequences for given proteins from neighboring taxa. Hundreds of predicted protein sequence database entries will be generated using bioinformatics based approaches such as sequence alignment, simple weighted consensus and point-assisted mutation matrices and automated for shareware on the web. Novel peptide sequences will be discovered through unambiguous matching of tandem mass spectra against the predicted protein sequences using database-scoring algorithms for mass spectrometry based proteomics such as Sequest. Novel sequences, unique to the taxon in question, will be rigorously validated by score cutoffs and manual inspection and then by synthesizing the resulting putative peptides for tandem mass spectral analysis as well as RNA sequencing of the test taxon, if possible. The analysis will be developed for several modern unsequenced taxa, including ostrich and alligator. The method will ultimately be used to sequence unique peptides from the soft tissue of the 68 million-year old Tyrannosaurus rex fossil found in Montana in 2003. This work will NOT (and in fact, cannot) result in the complete genetic sequencing of T. rex - we do not have a full set of T. rex proteins (the proteome), and if peptides have survived, they are separated from actual DNA code by levels of transcription and translation that are truly lost in time.The advantage of this new approach to fossils is that very low levels of proteinaceous material, including modified proteins, can be sequenced in a high-throughput manner with highly sensitive and fast-scanning mass spectrometers. The method can be used as a means of accurately identifying the organisms of fossils once a database of many unique protein sequences has been compiled. It will also help researchers with non-extinct organisms whose genomic content has not been sequenced, and will contribute a concrete test of assumptions made about molecular clocks in peptides and proteins. In addition, examination of the fossil materials will open a new window into molecular-level taphonomy, and establish a precedent for the study of molecular phylogeny in dinosaurs.