Large-scale characterization of HeLa cell nuclear phosphoproteins

被引:1144
作者
Beausoleil, SA
Jedrychowski, M
Schwartz, D
Elias, JE
Villén, J
Li, JX
Cohn, MA
Cantley, LC
Gygi, SP
机构
[1] Harvard Univ, Sch Med, Dept Syst Biol, Boston, MA 02115 USA
[2] Harvard Univ, Sch Med, Dept Cell Biol, Boston, MA 02115 USA
[3] Harvard Univ, Sch Med, Taplin Biol Mass Spectrometry Facil, Boston, MA 02115 USA
[4] Harvard Univ, Sch Med, Dana Farber Canc Inst, Boston, MA 02115 USA
关键词
phosphorylation; mass spectrometry; strong cation exchange chromatography;
D O I
10.1073/pnas.0404720101
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:12130 / 12135
页数:6
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