MudPIT analyses of the human proteins biotinylated by proximity to biotin ligase (BioID) fused to the C-terminus of activators of the dynein/dynactin motor

ABSTRACT: 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.

INSTRUMENT(S): LTQ

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

SUBMITTER: Laurence Florens  

PROVIDER: MSV000080808 | MassIVE | Thu Mar 30 10:58:00 BST 2017

SECONDARY ACCESSION(S): PXD006221

REPOSITORIES: MassIVE