Proteogenomic Analysis of C. elegans

描述（由申请人提供）：强大的基因组测序技术已经完成了180多个基因组，另外还有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。我们建议用高通量质谱法对复杂动物秀丽隐杆线虫的整个蛋白质补体进行补充，以补充现有的基于信使rna的方法。我们的方法结合了开放阅读框（ORF）分析全测序秀丽隐杆线虫基因组与高通量质谱，使用多维蛋白鉴定技术（MudPIT）。我们的长期目标是发展这些方法，使新测序的生物体中至少80%的蛋白质可以在几个月的时间内由一小群研究人员共同努力鉴定出来。这一目标需要开发以下工具：1)基因组orf的高效进化分析，以确定一组计算可管理的候选肽，用于质谱匹配；2)从整个生物体或组织中分离完整蛋白质的可靠方法；3)评估MudPIT与特定候选肽匹配意义的分析方法。我们的实验室已经建立了肽裂解、分离和二维（2D）质谱分析方法，目前这些工具的加入足以实现我们的目标。在这2年的资助期内，我们的具体里程碑是鉴定出至少10,000种独特的蛋白质（约占秀丽隐杆线虫所有预测蛋白质的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。最终的结果将是线虫蛋白质组的广泛图谱和高通量管道，这将允许对任何复杂的动物或植物蛋白质组进行类似的分析，其基因组序列是可用的。

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.