title: An Optimized Fluorescence-Based Bidimensional Immunoproteomic Approach for Accurate Screening of Autoantibodies.
abstract: Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.
authors.affiliation: Institut d'Immunologie, Centre de Biologie Pathologie et Génétique, Centre Hospitalier Régional Universitaire, Lille, France; UMR 995, LIRIC, INSERM, Lille, France; EA 2686, Univ Lille Nord de France, Lille, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Dutoit-Lefèvre
authors.affiliation: Institut d'Immunologie, Centre de Biologie Pathologie et Génétique, Centre Hospitalier Régional Universitaire, Lille, France; UMR 995, LIRIC, INSERM, Lille, France; EA 2686, Univ Lille Nord de France, Lille, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Dubucquoi
authors.affiliation: UMR 995, LIRIC, INSERM, Lille, France; EA 2686, Univ Lille Nord de France, Lille, France; Service de Médecine Interne et d'Immunologie clinique, Centre National de Référence Maladies rares, Centre Hospitalier Régional Universitaire, Lille, France.
authors.forename: David
authors.initials: D
authors.lastname: Launay
authors.affiliation: UMR 995, LIRIC, INSERM, Lille, France; EA 2686, Univ Lille Nord de France, Lille, France; Service de Médecine Interne et d'Immunologie clinique, Centre National de Référence Maladies rares, Centre Hospitalier Régional Universitaire, Lille, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Sobanski
authors.affiliation: Institut d'Immunologie, Centre de Biologie Pathologie et Génétique, Centre Hospitalier Régional Universitaire, Lille, France; UMR 995, LIRIC, INSERM, Lille, France; EA 2686, Univ Lille Nord de France, Lille, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Dussart
authors.affiliation: INSERM U1016, Institut Cochin, Paris, France; UMR 8104, CNRS Paris, France; Plate-forme Protéomique 3P5, Univ Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Chafey
authors.affiliation: INSERM U1016, Institut Cochin, Paris, France; UMR 8104, CNRS Paris, France; Plate-forme Protéomique 3P5, Univ Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Broussard
authors.affiliation: EA 2686, Univ Lille Nord de France, Lille, France; LBHE, EA 2465, UArtois, Lens, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Duban-Deweer
authors.affiliation: UMR 995, LIRIC, INSERM, Lille, France; EA 2686, Univ Lille Nord de France, Lille, France; Service de Neurologie D, Centre Hospitalier Régional Universitaire, Lille, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Vermersch
authors.affiliation: Institut d'Immunologie, Centre de Biologie Pathologie et Génétique, Centre Hospitalier Régional Universitaire, Lille, France; UMR 995, LIRIC, INSERM, Lille, France; EA 2686, Univ Lille Nord de France, Lille, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Prin
authors.affiliation: UMR 995, LIRIC, INSERM, Lille, France; EA 2686, Univ Lille Nord de France, Lille, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Lefranc
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000276:immunology*; D015153:Blotting, Western / Q000379:methods*; D002232:Carbocyanines / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis*; D056945:Hep G2 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007074:Immunoglobulin G / Q000097:blood; D007525:Isoelectric Focusing; D008163:Luminescent Measurements; D008180:Lupus Erythematosus, Systemic / Q000097:blood / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D040901:Proteomics / Q000379:methods*; D057825:Two-Dimensional Difference Gel Electrophoresis / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D002232:Carbocyanines; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; C089187:cyanine dye 3; C085321:cyanine dye 5