Enrichment of Phosphoproteins for Proteomic Analysis Using Immobilized Fe(III)-Affinity Adsorption Chromatography

Ida Chiara Guerrera,† Jelena Predic-Atkinson,† Oliver Kleiner,† Vukic Soskic,‡ and Jasminka Godovac-Zimmermann†
Centre for Molecular Medicine, Department of Medicine, University College London, 5 University Street, WC1E 6JJ London, United Kingdom, and Proteosys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany
J. Proteome Res., 2005, 4 (5), pp 1545–1553
DOI: 10.1021/pr050098m
Publication Date (Web): August 20, 2005
Copyright © 2005 American Chemical Society
†

 University College London.

,
‡

 Proteosys AG.

Abstract

Abstract Image

We described an efficient protocol to strongly enrich phosphoproteins from mixtures of total cellular proteins using homemade, recyclable Fe(III)-affinity columns. An integral feature of the method is the use of a detergent cocktail that allows use of different pHs for total protein extraction (pH 6.8) and for subsequent affinity capture of phosphoproteins (pH 3.4). Affinity captured proteins from rat fibroblasts were fractionated on 2D gels and random selection was identified by mass spectrometry. More than 85% of identified proteins were previously known to be phosphorylated. The specificity of the method was further validated by isolating proteins from 32P labeled cells. Our comparison of the clusters of acidic residues in the captured proteins with acidic clusters in proteins of the rat genome indicates that affinity for phosphate groups dominates over adsorption of proteins with acidic clusters.

Keywords: proteomics • phosphoproteomics • affinity adsorption chromatography • post-translational modifications • signal transduction

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History

  • Published In Issue October 10, 2005
  • Received April 11, 2005

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