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

SGER：一种从未测序类群中对新肽进行测序的方法：6800 万年前霸王龙的软组织

• 批准号: 0634136
• 负责人: John Asara
• 金额: $ 11.27万
• 依托单位: Beth Israel Deaconess Medical Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-10-01 至 2008-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0634136&HistoricalAwards=false
• 关键词: SGER; Method; Sequence; Novel; Peptides

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.

这项探索性研究的小额赠款将允许对一种新的、基于生物信息学和质谱的从头肽测序方法进行初步研究，以对来自其蛋白质或基因组内容先前未被测序的分类群的保守蛋白质进行测序。该方法将提供分子系统发育假说，并可能证明古化石中的肽含量。这些信息将有助于阐明未知分类群的系统发育，并将为生物学和古生物学研究人员提供有用的信息。该策略依赖于数据库搜索的串联质谱的肽与假设产生的预测蛋白质序列的基础上已知序列的给定蛋白质从邻近类群。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，并自动在网络上共享软件。新的肽序列将通过使用基于质谱的蛋白质组学的数据库评分算法（如Sequest）将串联质谱与预测的蛋白质序列进行明确匹配来发现。新的序列，独特的分类单元的问题，将严格验证分数截断和人工检查，然后通过合成得到的推定肽串联质谱分析以及RNA测序的测试分类单元，如果可能的话。将对包括鸵鸟和短吻鳄在内的几个现代未测序分类群进行分析。该方法最终将用于对2003年在蒙大拿州发现的6800万年前的霸王龙雷克斯化石软组织中的独特肽进行测序。这项工作不会（事实上，也不能）导致T的完整基因测序。雷克斯-我们没有全套的T。雷克斯蛋白质（蛋白质组），如果肽存活下来，它们与实际的DNA代码是分开的转录和翻译水平，真正失去了时间。这种新的化石方法的优点是，非常低水平的蛋白质材料，包括修饰蛋白质，可以在高通量的方式测序与高灵敏度和快速扫描质谱仪。该方法可以用来作为一种手段，准确地确定生物化石一旦数据库中的许多独特的蛋白质序列已经编译。它还将帮助研究人员研究基因组内容尚未测序的非灭绝生物，并将有助于对肽和蛋白质中分子钟的假设进行具体测试。此外，对化石材料的研究将为分子水平的埋藏学打开一扇新的窗口，并为恐龙的分子埋藏学研究建立先例。