title: Biotin tagging coupled with amino acid-coded mass tagging for efficient and precise screening of interaction proteome in mammalian cells.
abstract: In mammalian cells, when tandem affinity purification approach is employed, the existence of untagged endogenous target protein and repetitive washing steps together result in overall low yield of purified/stable complexes and the loss of weakly and transiently interacting partners of biological significance. To avoid the trade-offs involving in methodological sensitivity, precision, and throughput, here we introduce an integrated method, biotin tagging coupled with amino acid-coded mass tagging, for highly sensitive and accurate screening of mammalian protein-protein interactions. Without the need of establishing a stable cell line, using a short peptide tag which could be specifically biotinylated in vivo, the biotin-tagged target/bait protein was then isolated along with its associates efficiently by streptavidin magnetic microbeads in a single step. In a pulled-down complex amino acid-coded mass tagging serves as "in-spectra" quantitative markers to distinguish those bait-specific interactors from non-specific background proteins under stringent criteria. Applying this biotin tagging coupled with amino acid-coded mass tagging approach, we first biotin-tagged in vivo a multi-functional protein family member, 14-3-3epsilon, which was expressed at close to endogenous level. Starting with approximately 20 millions of 293T cells which were significantly less than what needed for a tandem affinity purification run, 266 specific interactors of 14-3-3epsilon were identified in high confidence.
authors.affiliation: Department of Proteomics and System Biology, Institute of Biomedical Science, Fudan University, Shanghai, PR China.
authors.forename: Yu-Fei
authors.initials: YF
authors.lastname: He
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Bao
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Xiao
authors.forename: Shuai
authors.initials: S
authors.lastname: Zuo
authors.forename: Ru-Yun
authors.initials: RY
authors.lastname: Du
authors.forename: Si-Wei
authors.initials: SW
authors.lastname: Tang
authors.forename: Peng-Yuan
authors.initials: PY
authors.lastname: Yang
authors.forename: Xian
authors.initials: X
authors.lastname: Chen
mesh_terms: D048948:14-3-3 Proteins / Q000032:analysis* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008280:Magnetics; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D019809:Streptavidin / Q000378:metabolism; D014162:Transfection
chemical_list: D048948:14-3-3 Proteins; D020543:Proteome; D001710:Biotin; D019809:Streptavidin