title: Identification of metallothionein subisoforms in HPLC using accurate mass and online sequencing by electrospray hybrid linear ion trap-orbital ion trap mass spectrometry.
abstract: A comprehensive approach to the characterization of metallothionein (MT) isoforms based on microbore HPLC with multimodal detection was developed. MTs were separated as Cd(7) complexes, detected by ICP MS and tentatively identified by molecular mass measured with 1-2 ppm accuracy using Orbital ion trap mass spectrometry. The identification was validated by accurate mass of the corresponding apo-MTs after postcolumn acidification and by their sequences acquired online by higher-energy collision dissociation MS/MS. The detection limits down to 10 fmol and 45 fmol could be obtained by ESI MS for apo- and Cd(7)-isoforms, respectively, and were lower than those obtained by ICP MS (100 fmol). The individual MT isoforms could be sequenced at levels as low as 200 fmol with the sequence coverage exceeding 90%. The approach was successfully applied to the identification of MT isoforms induced in a pig kidney cell line (LLC-PK(1)) exposed to CdS nanoparticles.
authors.affiliation: CNRS/UPPA, Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, UMR 5254, 2, av. Pr. Angot, 64053 Pau, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Mounicou
authors.forename: Laurent
authors.initials: L
authors.lastname: Ouerdane
authors.forename: Béatrice
authors.initials: B
authors.lastname: L'Azou
authors.forename: Isabelle
authors.initials: I
authors.lastname: Passagne
authors.forename: Céline
authors.initials: C
authors.lastname: Ohayon-Courtès
authors.forename: Joanna
authors.initials: J
authors.lastname: Szpunar
authors.forename: Ryszard
authors.initials: R
authors.lastname: Lobinski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008668:Metallothionein / Q000032:analysis*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000032:analysis; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013552:Swine
chemical_list: D020033:Protein Isoforms; D008668:Metallothionein