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Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12130-5. Epub 2004 Aug 9.

Large-scale characterization of HeLa cell nuclear phosphoproteins.

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  • 1Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

Abstract

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.

PMID:
15302935
[PubMed - indexed for MEDLINE]
PMCID:
PMC514446
Free PMC Article

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