Dataset Information

MudPIT analyses of the human proteins biotinylated by proximity to dynein/dynactin subunits fused to biotin ligase (BioID)

ABSTRACT: We identified the human dynein proteome by attaching a promiscuous biotin ligase (BioID) to 7 distinct components of the dynein machinery, including a subunit of its essential cofactor dynactin. BioID experiments were performed with stable HEK293 cell lines expressing dynein (IC1, IC2, LIC1, LIC2, RB, TcTex-1) and dynactin (p62) subunits tagged with BioID-3X FLAG. BioID-only transfected cells were used as negative controls. For BioID experiments, cells were lysed in the presence of detergents to disrupt the dynein/dynactin complex, allowing the identification of proteins that were proximal to the tagged subunit prior to cell lysis. After purification of biotinylated proteins on streptavidin-conjugated beads, the eluates were precipitated with TCA. After washing with acetone, the protein mixtures were digested with endoproteinase Lys-C and trypsin (Roche) and analyzed by MudPIT. We performed BioID purification followed by MudPIT in quadruplicate and used a label-free quantitative proteomics approach to calculate the enrichment of each identified protein relative to BioID control replicates.

INSTRUMENT(S): LTQ

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Laurence Florens