To understand how a single motor achieves cargo specificity, we attached a promiscuous biotin ligase (BioID) to 6 distinct activators of the dynein machinery motile activity, including two new activators, ninein and ninein-like. BioID experiments were performed with stable HEK293 cell lines expressing full length BICD1, BICD2, HOOK1, HOOK3, NIN and NINL with BioID tags at their C-termini. 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 activator 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 (Promega) 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.