UNIT 13.9 Phosphopeptide Mapping and Identification of Phosphorylation Sites

  1. Jill Meisenhelder1,
  2. Tony Hunter1,
  3. Peter van der Geer2

Published Online: 1 MAY 2001

DOI: 10.1002/0471140864.ps1309s18

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Meisenhelder, J., Hunter, T. and van der Geer, P. 2001. Phosphopeptide Mapping and Identification of Phosphorylation Sites. Current Protocols in Protein Science. 18:13.9:13.9.1–13.9.27.

Author Information

  1. 1

    The Salk Institute for Biological Studies, La Jolla, California

  2. 2

    University of California, San Diego, La Jolla, California

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: DEC 1999


Protein phosphorylation is a common modification for many proteins in the cell. Phosphorylation can affect localization of a protein, its stability, and its ability to dimerize or form stable complexes with other molecules. To understand the underlying mechanisms behind the phosphorylation of a given protein, it is often necessary to precisely identify which amino acid residues are phosphorylated. This unit describes the technique of phosphopeptide mapping. In this procedure, a radiolabeled protein is proteolytically digested, and the resulting phosphopeptides are separated in two dimensions on a TLC plate. The phosphopeptides are also analyzed by HPLC and mass spectrometry or peptide microsequencing. Such mapping gives information about the number of phosphorylation sites present in the protein, and can also be used to find out if sites of phosphorylation on a protein change upon treatment of cells with specific agents.