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.
authors.affiliation: Department of Chemistry, University of York, York, UK.
authors.forename: Barbara
authors.initials: B
authors.lastname: Pioselli
authors.forename: Caroline
authors.initials: C
authors.lastname: Munro
authors.forename: Andrea
authors.initials: A
authors.lastname: Raab
authors.forename: Christian L
authors.initials: CL
authors.lastname: Deitrich
authors.forename: Kriangsak
authors.initials: K
authors.lastname: Songsrirote
authors.forename: Jörg
authors.initials: J
authors.lastname: Feldmann
authors.forename: Jane
authors.initials: J
authors.lastname: Thomas-Oates
mesh_terms: D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003300:Copper / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D008099:Liver / Q000737:chemistry; D013058:Mass Spectrometry; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism; D012756:Sheep; D015032:Zinc / Q000737:chemistry
chemical_list: D008667:Metalloproteins; D020543:Proteome; D003300:Copper; D015032:Zinc