title: Characterization of a Protein Interactome by Co-Immunoprecipitation and Shotgun Mass Spectrometry.
abstract: Identifying the partners of a given protein (the interactome) may provide leads about the protein's function and the molecular mechanisms in which it is involved. One of the alternative strategies used to characterize protein interactomes consists of co-immunoprecipitation (co-IP) followed by shotgun mass spectrometry. This enables the isolation and identification of a protein target in its native state and its interactome from cells or tissue lysates under physiological conditions. In this chapter, we describe a co-IP protocol for interactome studies that uses an antibody against a protein of interest bound to protein A/G plus agarose beads to isolate a protein complex. The interacting proteins may be further fractionated by SDS-PAGE, followed by in-gel tryptic digestion and nano liquid chromatography high-resolution tandem mass spectrometry (nLC ESI-MS/MS) for identification purposes. The computational tools, strategy for protein identification, and use of interactome databases also will be described.
authors.affiliation: Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Munich, Germany.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Maccarrone
authors.affiliation: Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA), CONICET, Partner Institute of the Max Planck Society, Buenos Aires, Argentina.
authors.forename: Juan Jose
authors.initials: JJ
authors.lastname: Bonfiglio
authors.affiliation: Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA), CONICET, Partner Institute of the Max Planck Society, Buenos Aires, Argentina.
authors.forename: Susana
authors.initials: S
authors.lastname: Silberstein
authors.affiliation: Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Munich, Germany.
authors.forename: Christoph W
authors.initials: CW
authors.lastname: Turck
authors.affiliation: Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Rua Monteiro Lobato 255, 13083-862, Campinas, SP, Brazil. dmsouza@unicamp.br.|UNICAMP's Neurobiology Center, Campinas, Brazil. dmsouza@unicamp.br.
authors.forename: Daniel
authors.initials: D
authors.lastname: Martins-de-Souza
mesh_terms: D002460:Cell Line; D019295:Computational Biology / Q000379:methods; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
keywords: Co-immunoprecipitation; Computational tools; Interactome; Mass spectrometry; Proteome