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.