title: Speciation analysis for trace levels of selenoproteins in cultured human cells.
abstract: 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).
  
  
  CONCLUSIONS
  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 0.06pH 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.
authors.affiliation: CNRS/UPPA, Laboratoire de Chimie Analytique Bio-inorganique et Environnement (LCABIE), UMR5254, Hélioparc, 2, Av. Pr. Angot, 64053 Pau, France.
authors.forename: Juliusz
authors.initials: J
authors.lastname: Bianga
authors.affiliation: CNRS, UPR 3404, FRC3115 - Centre de Génétique Moléculaire, 91198 Gif-sur-Yvette, France.
authors.forename: Zahia
authors.initials: Z
authors.lastname: Touat-Hamici
authors.affiliation: CNRS/UPPA, Laboratoire de Chimie Analytique Bio-inorganique et Environnement (LCABIE), UMR5254, Hélioparc, 2, Av. Pr. Angot, 64053 Pau, France.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Bierla
authors.affiliation: CNRS/UPPA, Laboratoire de Chimie Analytique Bio-inorganique et Environnement (LCABIE), UMR5254, Hélioparc, 2, Av. Pr. Angot, 64053 Pau, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Mounicou
authors.affiliation: CNRS/UPPA, Laboratoire de Chimie Analytique Bio-inorganique et Environnement (LCABIE), UMR5254, Hélioparc, 2, Av. Pr. Angot, 64053 Pau, France. Electronic address: joanna.szpunar@univ-pau.fr.
authors.forename: Joanna
authors.initials: J
authors.lastname: Szpunar
authors.affiliation: CNRS/UPPA, Laboratoire de Chimie Analytique Bio-inorganique et Environnement (LCABIE), UMR5254, Hélioparc, 2, Av. Pr. Angot, 64053 Pau, France; CNRS, UPR 3404, FRC3115 - Centre de Génétique Moléculaire, 91198 Gif-sur-Yvette, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Chavatte
authors.affiliation: CNRS/UPPA, Laboratoire de Chimie Analytique Bio-inorganique et Environnement (LCABIE), UMR5254, Hélioparc, 2, Av. Pr. Angot, 64053 Pau, France; Department of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland.
authors.forename: Ryszard
authors.initials: R
authors.lastname: Lobinski
mesh_terms: D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D005260:Female; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D008297:Male; D040901:Proteomics / Q000379:methods*; D051140:Selenoproteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D051140:Selenoproteins
keywords: Cell line; ESI Orbitrap MS/MS; ICP MS; Laser ablation; Selenoprotein