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Phosphospecific proteolysis for mapping sites of protein phosphorylation

Nature Biotechnology volume 21, pages 10471054 (2003) | Download Citation

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  • A Corrigendum to this article was published on 01 November 2003

Abstract

Protein phosphorylation is a dominant mechanism of information transfer in cells, and a major goal of current proteomic efforts is to generate a system-level map describing all the sites of protein phosphorylation. Recent efforts have focused on developing technologies for enriching and quantifying phosphopeptides. Identification of the sites of phosphorylation typically relies on tandem mass spectrometry to sequence individual peptides. Here we describe an approach for phosphopeptide mapping that makes it possible to interrogate a protein sequence directly with a protease that recognizes sites of phosphorylation. The key to this approach is the selective chemical transformation of phosphoserine and phosphothreonine residues into lysine analogs (aminoethylcysteine and β-methylaminoethylcysteine, respectively). Aminoethylcysteine-modified peptides are then cleaved with a lysine-specific protease to map sites of phosphorylation. A blocking step enables single-site cleavage, and adaptation of this reaction to the solid phase facilitates phosphopeptide enrichment and modification in one step.

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Acknowledgements

We thank Matt Simon and Dustin Maly for helpful advice. Z.A.K. is a Howard Hughes Medical Institute Predoctoral Fellow. K.M.S. acknowledges support from the National Institutes of Health (EB001987).

Author information

Affiliations

  1. Program in Chemistry and Chemical Biology, University of California, San Francisco, California, USA.

    • Zachary A Knight
    •  & Denise M Kenski
  2. The Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, California 94945, USA.

    • Birgit Schilling
    • , Richard H Row
    •  & Bradford W Gibson
  3. Department of Pharmaceutical Chemistry, University of California, Box 0446, San Francisco, California 94143-0446, USA.

    • Bradford W Gibson
  4. Department of Cellular and Molecular Pharmacology, Box 2280, University of California, San Francisco, California 94143-2280, USA.

    • Kevan M Shokat
  5. Department of Chemistry, University of California, Berkeley, California 94720-1460, USA.

    • Kevan M Shokat

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Kevan M Shokat.

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DOI

https://doi.org/10.1038/nbt863

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