Speciation analysis for trace levels of selenoproteins in cultured human cells Juliusz Bianga a , Zahia Touat-Hamici b , Katarzyna Bierla a , Sandra Mounicou a , Joanna Szpunar a, ⁎ , Laurent Chavatte a, b , Ryszard Lobinski a, c a CNRS/UPPA, Laboratoire de Chimie Analytique Bio-inorganique et Environnement (LCABIE), UMR5254, Hélioparc, 2, Av. Pr. Angot, 64053 Pau, France b CNRS, UPR 3404, FRC3115 — Centre de Génétique Moléculaire, 91198 Gif-sur-Yvette, France c Department of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland ARTICLE INFO ABSTRACT Article history: Received 7 April 2014 Accepted 27 May 2014 Available online 4 June 2014 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 0.06 pH unit) using 3–10 pI strips. The protein detection limits were down to 1 ng mL -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 75 Se. 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 0.06 pH unit) using 3–10 pI strips. The protein detection limits were down to 1 ng mL -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 Keywords: Selenoprotein Cell line Laser ablation ICP MS ESI Orbitrap MS/MS JOURNAL OF PROTEOMICS 108 (2014) 316 – 324 ⁎ Corresponding author. Tel.: +33 559 40 77 55; fax: +33 559 40 77 82. E-mail address: joanna.szpunar@univ-pau.fr (J. Szpunar). http://dx.doi.org/10.1016/j.jprot.2014.05.025 1874-3919/© 2014 Elsevier B.V. All rights reserved. Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/jprot