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

Cite this

Denaturing and non-denaturing microsolution isoelectric focussing to mine the metalloproteome. / Pioselli, Barbara; Munro, Caroline; Raab, Andrea; Deitrich, Christian Lutz; Songsrirote, Kriangsak; Feldmann, Jorg; Thomas-Oates, Jane.

In: Metallomics, Vol. 1, No. 6, 2009, p. 501-510.

Research output: Contribution to journalArticle

Pioselli, B, Munro, C, Raab, A, Deitrich, CL, Songsrirote, K, Feldmann, J & Thomas-Oates, J 2009, 'Denaturing and non-denaturing microsolution isoelectric focussing to mine the metalloproteome', Metallomics, vol. 1, no. 6, pp. 501-510. https://doi.org/10.1039/b903607e
Pioselli, Barbara ; Munro, Caroline ; Raab, Andrea ; Deitrich, Christian Lutz ; Songsrirote, Kriangsak ; Feldmann, Jorg ; Thomas-Oates, Jane. / Denaturing and non-denaturing microsolution isoelectric focussing to mine the metalloproteome. In: Metallomics. 2009 ; Vol. 1, No. 6. pp. 501-510.
@article{1ce2bc19024e413a87b69ee7613f2c3f,
title = "Denaturing and non-denaturing microsolution isoelectric focussing to mine the metalloproteome",
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.",
keywords = "plasma-mass spectrometry, solution isoelectrofocusing prior, gel-electrophoresis, proteome analysis, 2-dimensional electrophoresis, complex proteomes, prefractionation techniques, sample prefractionation, immobiline membranes, carrier ampholytes",
author = "Barbara Pioselli and Caroline Munro and Andrea Raab and Deitrich, {Christian Lutz} and Kriangsak Songsrirote and Jorg Feldmann and Jane Thomas-Oates",
year = "2009",
doi = "10.1039/b903607e",
language = "English",
volume = "1",
pages = "501--510",
journal = "Metallomics",
issn = "1756-5901",
publisher = "Royal Society of Chemistry",
number = "6",

}

TY - JOUR

T1 - Denaturing and non-denaturing microsolution isoelectric focussing to mine the metalloproteome

AU - Pioselli, Barbara

AU - Munro, Caroline

AU - Raab, Andrea

AU - Deitrich, Christian Lutz

AU - Songsrirote, Kriangsak

AU - Feldmann, Jorg

AU - Thomas-Oates, Jane

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

KW - plasma-mass spectrometry

KW - solution isoelectrofocusing prior

KW - gel-electrophoresis

KW - proteome analysis

KW - 2-dimensional electrophoresis

KW - complex proteomes

KW - prefractionation techniques

KW - sample prefractionation

KW - immobiline membranes

KW - carrier ampholytes

U2 - 10.1039/b903607e

DO - 10.1039/b903607e

M3 - Article

VL - 1

SP - 501

EP - 510

JO - Metallomics

JF - Metallomics

SN - 1756-5901

IS - 6

ER -