title: Parallel Exploration of Interaction Space by BioID and Affinity Purification Coupled to Mass Spectrometry.
abstract: Complete understanding of cellular function requires knowledge of the composition and dynamics of protein interaction networks, the importance of which spans all molecular cell biology fields. Mass spectrometry-based proteomics approaches are instrumental in this process, with affinity purification coupled to mass spectrometry (AP-MS) now widely used for defining interaction landscapes. Traditional AP-MS methods are well suited to providing information regarding the temporal aspects of soluble protein-protein interactions, but the requirement to maintain protein-protein interactions during cell lysis and AP means that both weak-affinity interactions and spatial information is lost. A more recently developed method called BioID employs the expression of bait proteins fused to a nonspecific biotin ligase, BirA*, that induces in vivo biotinylation of proximal proteins. Coupling this method to biotin affinity enrichment and mass spectrometry negates many of the solubility and interaction strength issues inherent in traditional AP-MS methods, and provides unparalleled spatial context for protein interactions. Here we describe the parallel implementation of both BioID and FLAG AP-MS allowing simultaneous exploration of both spatial and temporal aspects of protein interaction networks.
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Geoffrey G
authors.initials: GG
authors.lastname: Hesketh
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Youn
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Payman
authors.initials: P
authors.lastname: Samavarchi-Tehrani
authors.affiliation: Princess Margaret Cancer Centre, Princess Margaret Research Institute, University Health Network, Toronto, Canada.|Department of Medical Biophysics, University of Toronto, Toronto, Canada.
authors.forename: Brian
authors.initials: B
authors.lastname: Raught
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada. gingras@lunenfeld.ca.|Department of Molecular Genetics, University of Toronto, Toronto, Canada. gingras@lunenfeld.ca.|Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Ave., Rm 992A (9th floor), Toronto, ON, Canada, M5G 1X5. gingras@lunenfeld.ca.
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gingras
mesh_terms: D019921:Biotinylation; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013223:Statistics as Topic; D057188:Workflow
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins
keywords: Affinity purification; BioID; Biotin; FLAG tag; Mass spectrometry; Protein identification; Protein interactions; Protein network; Proteomics; Proximity labeling; Streptavidin