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，另有700多个生物体的测序项目正在进行中。通过实验确定由这些基因组编码的蛋白质的困难和重要问题远远落后。我们建议补充现有的信使RNA为基础的方法与高通量质谱的整个蛋白质的一个复杂的动物，线虫秀丽隐杆线虫的补充。我们的方法结合了完整测序的C.利用多维蛋白质鉴定技术（MudPIT），通过高通量质谱技术对线虫基因组进行分析。我们的长期目标是发展这些方法，使一小群研究人员在几个月的共同努力下，可以鉴定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）基因组ORF的有效进化分析，以识别用于质谱匹配的计算可管理的候选肽组; 2）用于从整个生物体或组织中对完整蛋白质进行生化分级分离的稳健方法;以及3）评估MudPIT匹配特定候选肽的意义的分析方法。肽裂解，分馏，和二维（2D）质谱法是建立在我们的实验室，目前足以实现我们的目标，加上这些工具。在这两年的资助期内，我们的具体里程碑是鉴定出至少10，000种独特的蛋白质（占C. elegans），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。最终的结果将是一个广泛的C。elegans蛋白质组和一个高通量的管道，将允许类似的分析任何复杂的动物或植物蛋白质组的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.