TARGETED PROTEOMIC STUDY OF THE CYCLIN-CDK MODULE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 芽殖酵母酿酒酵母的细胞分裂周期主要由一种Cdk（细胞周期蛋白依赖性激酶）驱动，当与九种细胞周期蛋白亚基之一结合时，Cdk变得活跃。大多数特定的联营公司和这些细胞周期蛋白-Cdk复合物的基板仍然难以捉摸，虽然他们的澄清是必不可少的细胞周期的全面了解。 在这里，我们报告的结果，有针对性的蛋白质组学研究，确定了许多蛋白质与特定的细胞周期蛋白-Cdk复合物。 这些包括磷酸化底物，参与泛素化降解途径的蛋白质，衔接蛋白和抑制剂。我们研究并证实了其中一些相互作用的特异性和生物学相关性。 我们证明，这种方法用于研究蛋白质协会内的生物模块允许检测到许多新的协会。 我们的数据包括许多在以前的蛋白质组研究中遗漏的关联，并表明即使是瞬时和动态的相互作用也可以通过基于质谱的靶向蛋白质组学方法检测到。这项工作发表在Archambault V，Chang EJ，Drapkin BJ，Cross FR，Chait BT，Rout MP，Targeted Proteomic study of the cyclin-Cdk module Mol Cell. 2004，14，699-711。我们目前正在重新审视这个实验，在过去几年中，磷酸化富集程序，以及使用时间分辨蛋白质组学，优化的质谱读数，以及重同位素标记策略的高度优化的条件下。在这些新的条件下，我们已经分离出额外的假定底物，我们现在正在进行生物学测试。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The cell division cycle of the budding yeast Saccharomyces cerevisiae is mainly driven by one Cdk (Cyclin-Dependent Kinase), which becomes active when bound to one of nine cyclin subunits. The majority of the specific associates and substrates for these cyclin-Cdk complexes remain elusive, though their elucidation is essential for a full understanding of the cell cycle. Here we report the results of a targeted proteomics study that identified numerous proteins associated with particular cyclin-Cdk complexes. These included phosphorylation substrates, proteins involved in the ubiquitination-degradation pathway, adaptor proteins and inhibitors. We investigated and confirmed the specificity and biological relevance of some of these interactions. We demonstrate that this approach for studying protein associations within a biological module allowed the detection of many new associations. Our data includes many associations that were missed in previous proteome-wide studies, and shows that even transient and dynamic interactions can be detected by mass spectrometry-based targeted proteomic approaches. This work was presented in Archambault V, Chang EJ, Drapkin BJ, Cross FR, Chait BT, Rout MP, Targeted proteomic study of the cyclin-Cdk module Mol Cell. 2004, 14, 699-711. We are currently revisiting this experiment under conditions that have been highly optimized over the past several years, phosphorylation enrichment procedures, as well as using time-resolved proteomics, optimized mass spectrometric readout, together with heavy isotope labeling strategies. Under these new conditions, we have isolated additional putative substrates, which we are now testing biologically.