Speciation analysis for trace levels of selenoproteins in cultured human cells

J. Bianga , Z. Touat-Hamici , Katarzyna Bierla (1) , Sandra Mounicou (1) , Joanna Szpunar (1) , Chavatte Laurent (1) , Ryszard Lobinski (1)

1 IPREM - Institut des sciences analytiques et de physico-chimie pour l'environnement et les materiaux

A semi-quantitative method was developed for the non-targeted detection of trace levels of human selenoproteins in cytoplasmic cell extracts without the use of radioactive isotopes. The method was based on the direct detection of selenoproteins in iso-electrofocusing (IEF) electrophoretic strips by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS). The proteins were identified in the non-ablated parts of the gel corresponding to the LA-ICP MS peak apexes by electrospray Orbitrap MS/MS. The method allowed a high resolution of the selenoproteins (peak width 0.06pH unit) using 3-10 pI strips. The protein detection limits were down to 1ngmL-1 (as Se). The method was applied to the selenoprotein speciation in different human cell lines: Hek293 (kidney), HepG2 (liver), HaCaT (skin) and LNCaP (prostate). The principal proteins found included Selenoprotein 15 (Sep15), Glutathione peroxidase 1 (GPx1) and Glutathione peroxidase 4 (GPx4) and Thioredoxin reductase 1 (TRxR1) and Thioredoxin reductase 2 (TRxR2). Biological significance: Our paper presents the development of a semi-quantitative method for the non-targeted detection of trace levels of human selenoproteins in cytoplasmic cell extracts; it offers a first comprehensive screening of the entire biological selenoproteomes expressed in cell lines without the use of radioactive 75Se. The method was based on the direct detection of selenoproteins in iso-electrofocusing (IEF) electrophoretic strips by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS). The proteins were identified in the non-ablated parts of the gel corresponding to the LA-ICP MS peak apexes by electrospray Orbitrap MS/MS. The method allowed a high resolution of the selenoproteins (peak width 0.06pH unit) using 3-10 pI strips. The protein detection limits were down to 1ngmL-1 (as Se); by far the lowest ever reported. The method was applied to the selenoprotein speciation in different human cell lines: Hek293 (kidney), HepG2 (liver), HaCaT (skin) and LNCaP (prostate). The principal proteins found included Selenoprotein 15 (Sep15), Glutathione peroxidase 1 (GPx1) and Glutathione peroxidase 4 (GPx4) and Thioredoxin reductase 1 (TRxR1) and Thioredoxin reductase 2 (TRxR2). The IEF-LA-ICPMS indicates the presence of multiple forms of some selenoproteins which are for the moment impossible to distinguish because of the similarity of the bottom-up, proteomics data sets.

Mots clés: Cell line; ESI Orbitrap MS/MS; ICP MS; Laser ablation; Selenoprotein

Domaines: Chimie

J. Bianga, Z. Touat-Hamici, Katarzyna Bierla, Sandra Mounicou, Joanna Szpunar, et al.. Speciation analysis for trace levels of selenoproteins in cultured human cells. Journal of Proteomics, 2014, 108, pp.316-324. ⟨10.1016/j.jprot.2014.05.025⟩. ⟨hal-01505932⟩