title: A comparative study of the Se/S substitution in methionine and cysteine in Se-enriched yeast using an inductively coupled plasma mass spectrometry (ICP MS)-assisted proteomics approach.
abstract: A proteomics approach based on 2D gel electrophoresis followed by HPLC-electrospray Orbitrap MS/MS was developed to investigate the replacement and the degree of the Se/S substitution in methionine and cysteine in Se-rich yeast. Capillary HPLC-inductively coupled plasma mass spectrometry (ICP-MS), employed in parallel to capHPLC-ESI MS, indicated the virtual independence of the ESI MS response of the peptide structure (in the elution range of 30-65% methanol), and hence, the use of ESI MS data to determine the SeCys/Cys and SeMet/Met substitution ratios. For the first time a considerable incorporation of selenocysteine (SeCys) in proteins of the yeast proteome despite the absence of the UGA codon was demonstrated. The SeMet/Met and SeCys/Cys ratios were determined in a large number of peptides (57 and 26, respectively) issued from the tryptic digestion of 19 Se-containing proteins located in the gel by laser ablation-ICP MS imaging. The average Se/S substitution in methionine was 42.9±35.0 and was protein dependent with ratios ranging from 5 to 160 for individual peptides. The substitution of sulphur in cysteine (14.1±4.8%) in the cysteine-containing peptides was relatively similar (ratios from 9 to 23). Taking into account that the cysteine/methionine average ratio (2:1) in the yeast protein fraction, the study allowed the conclusion that 10-15% of selenium present in Se-enriched yeast is in the form of selenocysteine making up the mass balance of selenium species.
  
  
  CONCLUSIONS
  For the first time a considerable incorporation of selenocysteine (SeCys) in proteins of the yeast proteome despite the absence of the UGA codon was demonstrated. It was achieved using a proteomics approach based on 2D gel electrophoresis followed by HPLC-electrospray Orbitrap MS/MS in order to investigate the replacement and the degree of the Se/S substitution in methionine and cysteine in Se-rich yeast.
authors.affiliation: CNRS/UPPA, Laboratoire de Chimie Analytique Bio-inorganique et Environnement (LCABIE), UMR5254, Hélioparc, 2, av. Pr. Angot, 64053 Pau, France. katarzynabierla@wp.pl
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Bierla
authors.forename: Juliusz
authors.initials: J
authors.lastname: Bianga
authors.forename: Laurent
authors.initials: L
authors.lastname: Ouerdane
authors.forename: Joanna
authors.initials: J
authors.lastname: Szpunar
authors.forename: Alexandros
authors.initials: A
authors.lastname: Yiannikouris
authors.forename: Ryszard
authors.initials: R
authors.lastname: Lobinski
mesh_terms: D013058:Mass Spectrometry / Q000379:methods; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012643:Selenium / Q000378:metabolism; D017279:Selenocysteine / Q000235:genetics / Q000378:metabolism*; D012645:Selenomethionine / Q000378:metabolism*; D013455:Sulfur / Q000378:metabolism
chemical_list: D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D017279:Selenocysteine; D013455:Sulfur; D012645:Selenomethionine; D012643:Selenium