Proteogenomic Analysis of C. elegans

线虫的蛋白质组学分析

• 批准号: 7230204
• 负责人: JAMES H THOMAS
• 金额: $ 15.14万
• 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 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7230204
• 关键词: Animals; Applications Grants; Biochemical; C. elegans genome; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Code; Codon Nucleotides; Collaborations; Complement; Complex; Data; Detection; Development; Explosion; Fractionation; Gene Expression; Gene Structure; Genes; Genome; Genomics; Goals; Grant; Internet; Large-Scale Sequencing; Letters; Maps; Mass Spectrum Analysis; Messenger RNA; Methods; Molecular Genetics; Natural Selections; Nematoda; Open Reading Frames; Organism; Pattern; Peptides; Plants; Proteins; Proteome; Proteomics; RNA Splicing; Range; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Shotguns; Site; Techniques; Technology; Testing; Tissues; Validation; Whole Organism; base; computerized tools; experience; falls; genome sequencing; method development; tandem mass spectrometry; tool; two-dimensional

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已产生超过 180 个完整的基因组，另外 700 多个生物体的测序项目正在进行中。通过实验确定这些基因组编码的蛋白质这一困难而重要的问题远远落后。我们建议通过对复杂动物秀丽隐杆线虫的整个蛋白质进行高通量质谱分析来补充现有的基于信使 RNA 的方法。我们的方法使用多维蛋白质识别技术 (MudPIT)，将全测序线虫基因组的开放阅读框 (ORF) 分析与高通量质谱分析相结合。我们的长期目标是开发这些方法，让一小群研究人员在几个月的共同努力下，能够识别出新测序生物体中至少 80% 的蛋白质。这一目标需要开发以下工具：1）对基因组 ORF 进行有效的进化分析，以确定一组可计算管理的候选肽用于质谱匹配； 2）从整个生物体或组织中生化分离完整蛋白质的稳健方法； 3) 评估 MudPIT 与特定候选肽匹配的重要性的分析方法。我们的实验室建立了肽裂解、分级分离和二维 (2D) 质谱方法，目前通过添加这些工具足以实现我们的目标。我们在这两年资助期内的具体里程碑是鉴定至少 10,000 种独特蛋白质（> 线虫中所有预测蛋白质的 50%），并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。最终结果将是线虫蛋白质组的广泛图谱和高通量管道，该管道将允许对基因组序列可用的任何复杂动物或植物蛋白质组进行类似分析。