Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 33, Pages 12130-12135Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0404720101
Keywords
phosphorylation; mass spectrometry; strong cation exchange chromatography
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Funding
- NHGRI NIH HHS [HG00041, K22 HG000041, T32 HG000041] Funding Source: Medline
- NIGMS NIH HHS [R01 GM067945, GMS6203, R01 GM056203, GM67945] Funding Source: Medline
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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.
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