title: Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS.
abstract: Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by µbore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism.
authors: LCABIE - UMR5254, Technopôle Hélioparc Pau Pyrénées, 2 avenue du Président Angot, 64053 Pau Cedex 09, France.
authors: Ming
authors: M
authors: Xu
authors: IRSN/PRP-ENV/SERIS - Laboratoire de Biogéochimie, Biodisponibilité et Transferts des radionucléides, BP3, 13115 St. Paul-lez Durance Cedex, France.
authors: Sandrine
authors: S
authors: Frelon
authors: IRSN/PRP-ENV/SERIS - Laboratoire de Biogéochimie, Biodisponibilité et Transferts des radionucléides, BP3, 13115 St. Paul-lez Durance Cedex, France.
authors: Olivier
authors: O
authors: Simon
authors: LCABIE - UMR5254, Technopôle Hélioparc Pau Pyrénées, 2 avenue du Président Angot, 64053 Pau Cedex 09, France.
authors: Ryszard
authors: R
authors: Lobinski
authors: LCABIE - UMR5254, Technopôle Hélioparc Pau Pyrénées, 2 avenue du Président Angot, 64053 Pau Cedex 09, France. Electronic address: sandra.mounicou@univ-pau.fr.
authors: Sandra
authors: S
authors: Mounicou
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000032:analysis* / Q000737:chemistry; D003400:Astacoidea / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005293:Ferritins / Q000032:analysis / Q000737:chemistry; D005982:Glutathione Transferase / Q000032:analysis / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis / Q000737:chemistry; D043143:Hepatopancreas / Q000378:metabolism; D006657:Histones / Q000032:analysis / Q000737:chemistry; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods*; D011834:Radiation Monitoring / Q000379:methods; D015203:Reproducibility of Results; D013482:Superoxide Dismutase / Q000032:analysis / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods; D014305:Triose-Phosphate Isomerase / Q000032:analysis / Q000737:chemistry; D014501:Uranium / Q000032:analysis* / Q000737:chemistry
chemical_list: D060829:Arthropod Proteins; D006657:Histones; D014501:Uranium; D005293:Ferritins; D013482:Superoxide Dismutase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005982:Glutathione Transferase; D014305:Triose-Phosphate Isomerase
keywords: Gel electrophoresis; Identification; Laser ablation; Mass spectrometry; Protein; Uranium