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Non-denaturating isoelectric focusing gel electrophoresis for uranium-protein complexes quantitative analysis with LA-ICP MS

​Analytical and Bioanalytical Chemistry / February 2014, Volume 416, Issue 4, Pages 1063-1072

Document type > *Article de revue

Keywords >

Research Unit > IRSN/PRP-ENV/SERIS/L2BT

Authors > XU Ming, FRELON Sandrine, SIMON Olivier, LOBINSKI Ryszard, MOUNICOU Sandra

Publication Date > 01/02/2014

Summary

​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.


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