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.

这项用于探索性研究的小赠款将允许对新型，生物信息学和基于质谱的新颖肽测序方法进行初步研究，以从先前尚未对其蛋白质或基因组含量的分类群进行序列保守的蛋白质。该方法将提供分子系统发育假设，并有可能在古代化石中证明肽含量。该信息将有助于阐明未表征分类单元的系统发育，并将为生物学和古生物学研究人员提供有用的信息。该策略依赖于基于来自相邻分类单元的给定蛋白的已知序列的肽序列的串联质谱搜索数据库质谱。将使用基于生物信息学的方法（例如序列比对，简单加权共识和点辅助突变矩阵）生成数百种预测的蛋白质序列数据库条目，并在网络上进行共享软件自动化。新的肽序列将通过使用数据库量表算法（例如基于质谱的蛋白质组学（例如Sequest））与预测蛋白序列的明确匹配与预测的蛋白质序列进行明确匹配。新颖的序列是所讨论的分类单元独有的序列，将通过得分截止和手动检查，然后通过合成所得推定的肽进行串联质谱分析以及测试分类单元的RNA测序来严格验证。该分析将针对包括鸵鸟和鳄鱼在内的几个现代未序列分类单元进行开发。该方法最终将用于对2003年在蒙大拿州发现的6800万年历史的霸王龙化石的柔软组织进行独特的肽来序列。这项工作不会（实际上，实际上不能）导致T. rex的完整遗传测序 - 我们没有通过t. rex蛋白（如果是蛋白质）分离的，并且没有整体的蛋白质（pection and proce deciption and prective prote and pection and pection and pection and pection deciption depection and pection deciption depection and pecrive depection and pecription），并且是彼得彼得的，则是彼得的，并且是彼得的。这种新的化石方法的真正损失的翻译是，可以以高敏感和快速扫描的质谱仪的高通量方式对非常低的蛋白质材料（包括改良的蛋白质）进行测序。一旦收集了许多独特的蛋白质序列的数据库，该方法可以用作准确识别化石生物的一种手段。它还将帮助患有尚未对基因组含量的生物体的研究人员进行测序，并将对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级的taphonomy打开一个新窗口，并为研究恐龙中分子系统发育的先例建立了先例。