title: Immuno-proteomics: Development of a novel reagent for separating antibodies from their target proteins.
abstract: Immunoprecipitation (IP) is a widely used technique for identifying the binding partners of the target proteins of specific antibodies. Putative binding targets and their partners are usually in much lower amounts than the antibodies used to capture these target proteins. Thus antigen identification using proteomics following IP is often confounded by the presence of an overwhelming amount of interfering antibody protein. Even covalently linking antibodies to beads is susceptible to antibody leaching during IP. To circumvent this interference, we describe here a reagent, called Biotin-CDM that reversibly tags all potential target proteins in a cell lysate with biotin. The presence of biotin coupled to the target proteins allows for a secondary separation step in which antibodies are washed away from the reversibly biotinylated target proteins by binding them to an Avidin-coupled matrix. The captured target proteins are released from the Avidin matrix by reversing the Biotin-CDM link, thus releasing a pool of target proteins ready for further proteomic analysis compatible with 2D-electrophoresis. Here, we describe the synthesis and characterization of Biotin-CDM. We also demonstrate Biotin-CDM's use for immunoprecipitation of a known antigen, as well as its use for capturing an array of proteins targeted by the autoantibodies found in the serum a patient suffering from rheumatoid arthritis. The use of this reagent allows one to combine immunoprecipitation and 2D-Difference gel electrophoresis, overcoming the current limitations of Serological Proteome Analysis (SERPA) in discovering autoantigens. This article is part of a Special Issue entitled: Medical Proteomics.
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Vinitha
authors.initials: V
authors.lastname: Ganesan
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Schmidt
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Raghunandan
authors.initials: R
authors.lastname: Avula
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Dagney
authors.initials: D
authors.lastname: Cooke
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Taylor
authors.initials: T
authors.lastname: Maggiacomo
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Lawton
authors.initials: L
authors.lastname: Tellin
authors.affiliation: Division of Rheumatology, Department of Medicine, University of Miami Miller School of Medicine, 1600 Northwest 10th Avenue, Miami, FL 33136, USA.
authors.forename: Dana P
authors.initials: DP
authors.lastname: Ascherman
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Marcel P
authors.initials: MP
authors.lastname: Bruchez
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA. Electronic address: minden@cmu.edu.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Minden
mesh_terms: D001172:Arthritis, Rheumatoid / Q000097:blood*; D001323:Autoantibodies / Q000737:chemistry*; D001324:Autoantigens / Q000097:blood*; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D040901:Proteomics / Q000379:methods*
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D001360:Avidin; D001710:Biotin
keywords: Difference gel electrophoresis (DIGE); Immuno-proteomics; Immunoprecipitation; Reversible biotin; Serological proteomics