title: Non-denaturating isoelectric focusing gel electrophoresis for uranium-protein complexes quantitative analysis with LA-ICP MS.
abstract: A non-denaturating isoelectric focusing (ND-IEF) gel electrophoresis protocol has been developed to study and identify uranium (U)-protein complexes with laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS) and electrospray ionization mass spectrometry (ESI-MS). The ND-IEF-LA-ICP MS methodology set-up was initiated using in vitro U-protein complex standards (i.e., U-bovine serum albumin and U-transferrin) allowing the assessment of U recovery to 64.4 ± 0.4 %. This methodology enabled the quantification of U-protein complexes at 9.03 ± 0.23, 15.27 ± 0.36, and 177.31 ± 25.51 nmol U L(-1) in digestive gland cytosols of the crayfish, Procambarus clarkii, exposed respectively to 0, 0.12, and 2.5 μmol of waterborne depleted U L(-1) during 10 days. ND-IEF-LA-ICP MS limit of detection was 19.3 pmol U L(-1). Elemental ICP MS signals obtained both in ND-IEF electropherograms and in size exclusion chromatograms of in vivo U-protein complexes revealed interactions between U- and Fe- and Cu-proteins. Moreover, three proteins (hemocyanin, pseudohemocyanin-2, and arginine kinase) out of 42 were identified as potential uranium targets in waterborne-exposed crayfish cytosols by microbore reversed phase chromatography coupled to molecular mass spectrometry (µRPC-ESI-MS/MS) after ND-IEF separation.
authors.affiliation: LCABIE-UMR5254, Technopôle Hélioparc Pau Pyrénées, 2 avenue du Président Angot, 64053, Pau Cedex 09, France.
authors.forename: Ming
authors.initials: M
authors.lastname: Xu
authors.forename: Sandrine
authors.initials: S
authors.lastname: Frelon
authors.forename: Olivier
authors.initials: O
authors.lastname: Simon
authors.forename: Ryszard
authors.initials: R
authors.lastname: Lobinski
authors.forename: Sandra
authors.initials: S
authors.lastname: Mounicou
mesh_terms: D000818:Animals; D003400:Astacoidea; D002417:Cattle; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014501:Uranium / Q000737:chemistry*
chemical_list: D011506:Proteins; D014501:Uranium