Large-scale characterization of HeLa cell nuclear phosphoproteins
Large-scale characterization of HeLa cell nuclear phosphoproteins
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DOI：
10.1073/pnas.0404720101
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发表时间：
2004-08-17
影响因子：
11.1
通讯作者：
Gygi, SP
中科院分区：
文献类型：
--
作者：
Beausoleil, SA;Jedrychowski, M;Gygi, SP
确定调节磷酸化事件的位置对于阐明特定的激酶-底物关系，为理解重要的信号通路，并最终深入了解多种疾病病理机制，往往是至关重要的。尽管人们对蛋白质的磷酸化调控机制进行了大量的研究，但如何有效、大规模地鉴定和表征磷酸化位点仍然是一个尚未解决的问题。在这份报告中，我们描述了现有技术在分离和鉴定磷酸化位点方面的应用。采用一种基于强阳离子交换层析的策略，从HeLa细胞裂解物的核部分中富集磷酸肽。从967个蛋白质中，串联MS确定了2,002个磷酸化位点。这一史无前例的大量位点集合使得对已知和未知的激酶基序和底物进行了详细的计算。
Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.