Large-scale phosphorylation analysis of α-factor-arrested Saccharomyces cerevisiae
Large-scale phosphorylation analysis of α-factor-arrested Saccharomyces cerevisiae
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DOI：
10.1021/pr060559j
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发表时间：
2007-03-01
影响因子：
4.4
通讯作者：
Gygi, Steve P.
中科院分区：
文献类型：
--
作者：
Li, Xue;Gerber, Scott A.;Gygi, Steve P.
蛋白质磷酸化对于许多细胞过程是必不可少的。磷蛋白的大规模分析继续提高我们理解这些过程的深度和速度。近年来，有效的磷蛋白和肽富集技术的发展以及质谱仪器的改进加强了磷蛋白质组学的研究，取得了前所未有的成就。在这里，我们描述了一个大规模的磷酸化分析α-因子被捕酵母。使用多维分离策略，包括制备型SDS-PAGE预分馏，在凝胶消化与胰蛋白酶，和固定化金属亲和色谱（IMAC）富集的磷酸肽，然后通过LC-MS/MS分析采用混合LTQ-Orbitrap质谱仪，我们能够编目的相当大一部分存在于酵母全细胞裂解液中的磷蛋白。这个分析产生了2288个非冗余磷酸化位点的985蛋白质的信心鉴定。模糊度评分（Ascore）算法用于确定整个数据集的部位定位的确定性。此外，数据集的大小允许使用Motif-X算法提取已知和新的激酶基序。最后，大量的信息素信号通路的成员被发现磷蛋白和讨论。
Protein phosphorylation is essential for numerous cellular processes. Large-scale profiling of phosphoproteins continues to enhance the depth and speed at which we understand these processes. The development of effective phosphoprotein and peptide enrichment techniques and improvements to mass spectrometric instrumentation have intensified phosphoproteomic research in recent years, leading to unprecedented achievements. Here, we describe a large-scale phosphorylation analysis of alpha-factor-arrested yeast. Using a multidimensional separation strategy involving preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and immobilized metal affinity chromatography (IMAC) enrichment of phosphopeptides, followed by LC-MS/MS analysis employing a hybrid LTQ-Orbitrap mass spectrometer, we were able to catalog a substantial portion of the phosphoproteins present in yeast whole-cell lysate. This analysis yielded the confident identification of 2288 nonredundant phosphorylation sites from 985 proteins. The ambiguity score (Ascore) algorithm was utilized to determine the certainty of site localization for the entire data set. In addition, the size of the data set permitted extraction of known and novel kinase motifs using the Motif-X algorithm. Finally, a large number of members of the pheromone signaling pathway were found as phosphoproteins and are discussed.