title: Identification of mammalian protein complexes by lentiviral-based affinity purification and mass spectrometry.
abstract: Protein complexes and protein-protein interactions (PPIs) are fundamental for most biological functions. Deciphering the extensive protein interaction networks that occur within cellular contexts has become a logical extension to the human genome project. Proteome-scale interactome analysis of mammalian systems requires efficient methods for accurately detecting PPIs with specific considerations for the intrinsic technical challenges of mammalian genome manipulation. In this chapter, we outline in detail an innovative lentiviral-based functional proteomic approach that can be used to rapidly characterize protein complexes from a broad range of mammalian cell lines. This method integrates the following key features: (1) lentiviral elements for efficient delivery of tagged constructs into mammalian cell lines; (2) site-specific Gateway™ recombination sites for easy cloning; (3) versatile epitope-tagging system for flexible affinity purification strategies; and (4) LC-MS-based protein identification using tandem mass spectrometry.
authors.affiliation: Banting and Best Department of Medical Research, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
authors.forename: Zuyao
authors.initials: Z
authors.lastname: Ni
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Olsen
authors.forename: Andrew
authors.initials: A
authors.lastname: Emili
authors.forename: Jack F
authors.initials: JF
authors.lastname: Greenblatt
mesh_terms: D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D011506:Proteins; D020543:Proteome