Denaturing and non-denaturing microsolution isoelectric focussing to mine the metalloproteome

Barbara Pioselli, Caroline Munro, Andrea Raab, Christian Lutz Deitrich, Kriangsak Songsrirote, Jorg Feldmann, Jane Thomas-Oates

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Metals bound to proteins play essential roles in living systems. Elements such as phosphorus, selenium and iodine are commonly covalently linked to proteins while others are non-covalently complexed. Thus, the identification and characterization of the metal-protein complexes require a careful hyphenation of techniques able to separate and detect the intact binding complexes with both high resolution and high sensitivity. This study has investigated for the first time the potential of microsolution isoelectric focussing to separate a mixture of metal-binding protein standards under well-established denaturing conditions and a novel non-denaturing separation protocol has also been developed. SEC-ICP-MS analysis was used to evaluate the ability of the two separation procedures to separate and maintain the integrity of standard metal-protein complexes. Microsolution isoelectric focussing under denaturing conditions separates the metalloprotein mixtures with high resolution, although the stability of the complexes is affected. Microsolution isoelectric focussing under our newly developed non-denaturing conditions shows a lower degree of resolution, although the stability of the metal-protein complexes is preserved. The applicability of the two procedures to a biological metalloproteome has also been evaluated.

Original languageEnglish
Pages (from-to)501-510
Number of pages10
JournalMetallomics
Volume1
Issue number6
DOIs
Publication statusPublished - 2009

Keywords

  • plasma-mass spectrometry
  • solution isoelectrofocusing prior
  • gel-electrophoresis
  • proteome analysis
  • 2-dimensional electrophoresis
  • complex proteomes
  • prefractionation techniques
  • sample prefractionation
  • immobiline membranes
  • carrier ampholytes

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