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

这笔用于探索性研究的小笔资金将使一种基于生物信息学和质谱学的新的从头测序方法的初步工作得以进行，该方法可以对其蛋白质或基因组含量以前从未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多肽含量。这些信息将有助于阐明未描述的分类群的系统发育，并将为生物学和古生物学研究人员提供有用的信息。该策略依赖于对多肽的串联质谱图与假设生成的预测蛋白质序列的数据库搜索，这些预测蛋白质序列是基于来自邻近类群的给定蛋白质的已知序列。将使用基于生物信息学的方法，如序列比对、简单加权共识和点辅助突变矩阵，生成数百个预测的蛋白质序列数据库条目，并自动在网络上共享软件。通过使用SEQUEST等基于质谱学的数据库评分算法将串联质谱图与预测的蛋白质序列进行明确的匹配，将发现新的多肽序列。新的序列，是所讨论的分类群所独有的，将通过得分截止点和人工检查，然后合成用于串联质谱分析的推定多肽，以及如果可能的话，对测试分类群进行RNA测序，严格验证。这项分析将针对几个现代未测序的分类群进行，包括鸵鸟和短吻鳄。该方法最终将被用于对2003年在蒙大拿州发现的6800万年前的暴龙霸王龙化石的软组织中的独特多肽进行测序。这项工作不会(事实上也不可能)导致霸王龙的完整基因测序--我们没有完整的霸王龙蛋白质(蛋白质组)，如果多肽存活下来，它们就会通过真正丢失的转录和翻译水平与实际的DNA密码分开。这种新的化石方法的优势是，可以用高灵敏度和快速扫描的质谱仪以高通量的方式对非常低水平的蛋白质类物质进行测序，包括修饰的蛋白质。一旦编制了许多独特蛋白质序列的数据库，这种方法就可以作为一种准确识别化石有机体的手段。它还将帮助研究基因组内容尚未测序的非灭绝生物的研究人员，并将有助于对关于多肽和蛋白质中分子时钟的假设进行具体测试。此外，对化石材料的检查将为分子水平的敲门学打开一扇新的窗口，并为恐龙分子系统学研究开创先例。

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.