Biotin tagging coupled with amino acid-coded mass tagging for efficient and precise screening of interaction proteome in mammalian cells.
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.
He
Yu-Fei
YF
Department of Proteomics and System Biology, Institute of Biomedical Science, Fudan University, Shanghai, PR China.
Bao
Hui-Min
HM
Xiao
Xiao-Feng
XF
Zuo
Shuai
S
Du
Ru-Yun
RY
Tang
Si-Wei
SW
Yang
Peng-Yuan
PY
Chen
Xian
X
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
D048948:14-3-3 Proteins; D020543:Proteome; D001710:Biotin; D019809:Streptavidin