title: On-line capillary isoelectric focusing hyphenated to native electrospray ionization mass spectrometry for the characterization of interferon-γ and variants.
abstract: The on-line hyphenation of Capillary IsoElectric Focusing (CIEF) with ElectroSpray Ionization Mass Spectrometry (ESI/MS) has been carried out in a non-denaturing detection mode at the CIEF-MS interface. This CIEF-MS coupling methodology relied on the use of 40% glycerol-water medium as anti-convective agent in the CE capillary and the addition of 10 mM ammonium acetate buffer, pH 5, as a volatile aqueous sheath liquid. These CIEF-MS coupling conditions allowed the characterization of the highly basic cytokine human interferon-gamma (IFN-γ) and its detection as a non-covalent homodimer (33,814.3 g mol(-1)) corresponding to the active form of this immune-regulatory protein. An experimental pI value of 9.95 was determined for the human IFN-γ homodimer in these conditions. The CIEF-MS analysis of several variants bearing punctual or deletion mutations within the two D1 and D2 basic clusters at the C-terminal end of IFN-γ revealed the different contribution of these domains to the charge properties of this heparan sulfate-binding protein.
authors.affiliation: CNRS, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (UMR 8587), Bld. François Mitterrand, F-91025, Evry, France. regis.daniel@univ-evry.fr.
authors.forename: Cédric
authors.initials: C
authors.lastname: Przybylski
authors.forename: Meriem
authors.initials: M
authors.lastname: Mokaddem
authors.forename: Mehdi
authors.initials: M
authors.lastname: Prull-Janssen
authors.forename: Els
authors.initials: E
authors.lastname: Saesen
authors.forename: Hugues
authors.initials: H
authors.lastname: Lortat-Jacob
authors.forename: Florence
authors.initials: F
authors.lastname: Gonnet
authors.forename: Anne
authors.initials: A
authors.lastname: Varenne
authors.forename: Régis
authors.initials: R
authors.lastname: Daniel
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis* / Q000235:genetics; D007525:Isoelectric Focusing / Q000379:methods*; D017384:Sequence Deletion / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D007371:Interferon-gamma; D000099166:Polyacrylamides; C573415:IFNG protein, human