Protein identification by mass spectrometry

BioID samples were processed for mass spectrometry as outlined by (27). Briefly, streptavidin beads were washed with 50 mM NH₄HCO₃ and resuspended in 50 mM NH₄HCO₃ containing 5 mM dithiothreitol and heated at 75°C for 10 min. Iodoacetamide was added to a final concentration of 10 mM to each sample followed by incubation in the dark at room temperature for 20 min. Afterward, 1 mM CaCl₂ and fresh 5 mM dithiothreitol were added and incubation continued overnight. The following morning, trifluoroacetic acid (TFA) was added to a final concentration of 0.5% (v/v). Beads were pelleted using a magnetic rack and the supernatant removed. A second elution of digested peptides with 0.5% TFA was performed and the supernatant pooled. To each sample, 1 μg of sequence-grade trypsin was added and incubated overnight at 37°C. For 3x-FLAG peptide eluted material, in-solution trypsin digests were performed by incubating samples with 1 μg of sequence-grade trypsin (Promega) at 37°C in 1× TBS followed by lyophilization until dry. Digested peptides were passed over ZipTips (Millipore) and eluted with 0.1% TFA/50% acetonitrile. Peptides were analyzed by an Orbitrap LTQ mass spectrometer (Thermo Scientific) with result searches performed using the program MASCOT (Matrix Science). To identify protein interactors, peptide counts for sample versus control were submitted to the Crapome web server (28). Proteins identified that had a fold change greater than two, and at least two peptides detected, were considered to be enriched.