To identify non-specific biotinylation events, HEK293 cells transfected with a plasmid carrying only the biotin ligase moiety were used as negative controls to BioID experiments. Cells were lysed in the presence of detergents. 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.