Unit

UNIT 18.13 Isolation of Phosphopeptides by Immobilized Metal Ion Affinity Chromatography

  1. Thomas Nühse1,
  2. Kebing Yu2,
  3. Arthur Salomon2

Published Online: 1 JAN 2007

DOI: 10.1002/0471142727.mb1813s77

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Nühse, T., Yu, K. and Salomon, A. 2007. Isolation of Phosphopeptides by Immobilized Metal Ion Affinity Chromatography. Current Protocols in Molecular Biology. 77:18.13:18.13.1–18.13.23.

Author Information

  1. 1

    University of Manchester, Manchester, United Kingdom

  2. 2

    Brown University, Providence, Rhode Island

Publication History

  1. Published Online: 1 JAN 2007
  2. Published Print: JAN 2007

Abstract

The identification of protein phosphorylation sites from cell-derived proteins is crucial to the understanding of signal transduction pathways. While determining the modified sites on individual proteins can present a significant challenge, recent progress in the rapid, large-scale identification of phosphopeptides from complex protein mixtures by combinations of affinity chromatography and mass spectrometry provides a powerful tool to decipher the phosphoproteome. A set of protocols is described for sample preparation, fractionation, immobilized metal ion affinity chromatography (IMAC), and mass spectrometric analysis of phosphorylation sites. Parts of the protocols can be combined in different ways to adapt to sample amounts, complexity, and available equipment. Up to thousands of unique phosphopeptides can be sequenced from a single sample in a day, revealing a unique snapshot of global cellular phosphorylation sites on proteins and facilitating in-depth study of the identified phosphoproteins.

Keywords:

  • phosphorylation;
  • phosphopeptide;
  • mass spectrometry;
  • proteomics;
  • signal transduction