MOTIF-SPECIFIC SAMPLING OF PHOSPHOPROTEOMES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 磷酸蛋白质组学是对被磷酸化修饰的蛋白质亚组分的靶向性研究，已成为研究细胞信号动力学不可缺少的工具。我们描述了一种基于氨基酸的Ba2+结合特性将磷酸蛋白质组和蛋白质组分析联系起来的方法。这项技术选择了不依赖于被分析系统的基序特异的磷酸肽。MudPit(多维识别技术)从低至250微克的蛋白质中识别出1037个沉淀的磷酸肽。为了扩大磷酸蛋白质组的覆盖范围，我们采集了具有三个Ba2+离子摩尔比的HeLa细胞核提取液。超过70%的已鉴定的磷蛋白的存在被它们的未修饰的多肽进一步证实。在异丙肾上腺素刺激HEK细胞后，我们从MAPK级联和AKAP信号枢纽中鉴定出越来越多的磷酸蛋白。我们量化了197种磷酸蛋白的蛋白质和磷酸化水平的变化，其中包括关键的激酶MAPK1。MAPK1的差异磷酸化与知识库的整合构建了与其作为典型通路串话中的节点的角色很好地关联的模块。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phosphoproteomics, the targeted study of a subfraction of the proteome which is modified by phosphorylation, has become an indispensable tool to study cell signaling dynamics. We described a methodology that linked phosphoproteome and proteome analysis based on Ba2+ binding properties of amino acids. This technology selected motif-specific phosphopeptides independent of the system under analysis. MudPIT (Multidimensional Identification Technology) identified 1037 precipitated phosphopeptides from as little as 250 microg of proteins. To extend coverage of the phosphoproteome, we sampled the nuclear extract of HeLa cells with three values of Ba2+ ions molarity. The presence of more than 70% of identified phosphoproteins was further substantiated by their nonmodified peptides. Upon isoproterenol stimulation of HEK cells, we identified an increasing number of phosphoproteins from MAPK cascades and AKAP signaling hubs. We quantified changes in both protein and phosphorylation levels of 197 phosphoproteins including a critical kinase, MAPK1. Integration of differential phosphorylation of MAPK1 with knowledge bases constructed modules that correlated well with its role as node in cross-talk of canonical pathways.