Proteogenomic Analysis of C. elegans

线虫的蛋白质组学分析

• 批准号: 7093849
• 负责人: JAMES H THOMAS
• 金额: $ 15.22万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7093849
• 关键词: Caenorhabditis elegans; biochemical evolution; computational biology; gene expression; genetic strain; helminth genetics; high throughput technology; mass spectrometry; messenger RNA; natural selections; polymerase chain reaction; proteomics; subcellular fractionation

DESCRIPTION (provided by applicant): Robust genome sequencing technology has resulted in over 180 completed genomes, with sequencing projects for an additional 700+ organisms in progress. The difficult and important problem of experimentally determining the proteins encoded by these genomes lags far behind. We propose to complement existing messenger-RNA based approaches with high-throughput mass spectrometry of the entire protein complement of a complex animal, the nematode Caenorhabditis elegans. Our approach combines open- reading-frame (ORF) analysis of the fully sequenced C. elegans genome with high-throughput mass spectrometry, using multidimensional protein identification technology (MudPIT). Our long-term goal is development of these methods to the point that at least 80% of all proteins in a newly sequenced organism can be identified in a few months of concerted effort by a small group of investigators. This goal requires development of the following tools: 1) efficient evolutionary analysis of genomic ORFs to identify a computationally manageable set of candidate peptides for mass spectrum matching; 2) a robust method for biochemical fractionation of intact proteins from whole organisms or tissues; and 3) analytical approaches to assessing the significance of MudPIT matches to specific candidate peptides. Peptide cleavage, fractionation, and 2-dimensional (2D) mass spectrometry methods are established in our labs and are currently sufficient to achieve our goal with the addition of these tools. Our specific milestone for this 2-year grant period is the identification of at least 10,000 unique proteins (>50% of all predicted proteins in C. elegans) and validation by orthogonal methods of at least 50 of the proteins that are not yet supported by other data. The end result will be both an extensive map of the C. elegans proteome and a high-throughput pipeline that will allow similar analysis of any complex animal or plant proteome whose genome sequence is available.

描述（由申请人提供）：强大的基因组测序技术导致超过180个完整的基因组，并进行了额外700多种生物的测序项目。实验确定这些基因组编码的蛋白质的困难和重要问题远远落后。我们建议与现有的基于Messenger-RNA的方法相辅相成，并通过复杂动物的整个蛋白质补体，线虫Caenorhabditis elegans的高通量质谱。我们的方法使用多维蛋白质鉴定技术（MUDPIT）结合了对秀丽隐杆线虫基因组的开放式框架分析（ORF）分析和高通量质谱。我们的长期目标是开发这些方法，以至于可以在几个月的一小群研究人员的努力中确定新测序有机体中至少80％的蛋白质。该目标需要开发以下工具：1）基因组ORF的有效进化分析，以确定一组可管理的候选肽集以进行质谱匹配； 2）一种可靠的方法，用于从整个生物或组织中完整蛋白质的生化分馏； 3）分析方法评估泥浆匹配与特定候选肽的重要性。在我们的实验室中建立了肽的裂解，分级和二维（2D）质谱方法，目前足以通过添加这些工具来实现我们的目标。我们在这个2年赠款期间的具体里程碑是鉴定至少10,000种独特的蛋白质（>秀丽隐杆线虫中所有预测的蛋白质的50％）和通过正交方法验证至少50种尚未得到其他数据支持的蛋白质。最终结果既是秀丽隐杆线虫蛋白质组的广泛图，又是高通量管道，它将允许对任何具有基因组序列的复杂动物或植物蛋白质组进行类似的分析。