BioID sample preparation and MS

Growth of cells and sample preparation for BioID experiments were performed as previously described with slight modifications (Redwine et al., 2017). Briefly, BioID-3xFLAG or KIF1C-BioID-3xFLAG cells were plated at ∼20% confluence in 15-cm dishes as four replicates, with each replicate consisting of 8 × 15-cm plates. After 24 h, biotin was added to the media to a final concentration of 50 µM, and the cells were allowed to grow for another 16 h. After decanting the media, cells were dislodged from each plate by pipetting with ice-cold 1× PBS. Cells were centrifuged at 1,000 × g for 2 min followed by two washes with ice-cold 1× PBS, and the cell pellets were resuspended and lysed in 16 ml RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% [vol/vol] NP-40, 0.5% [wt/vol] sodium deoxycholate, 0.1% [wt/vol] SDS, 1 mM DTT, and protease inhibitors [cOmplete Protease Inhibitor Cocktail; Roche]) by gentle rocking for 15 min at 4°C. The cell lysate was clarified via centrifugation at 66,000 × g for 30 min in a Ti70 rotor (Beckman Coulter) at 4°C. The clarified lysate was retrieved and combined with prewashed 0.8 ml streptavidin-conjugated beads (Pierce Streptavidin magnetic beads) and incubated overnight at 4°C with gentle rocking. Bead/lysate mixtures were collected on a magnetic stand into a single 2-ml round-bottom microcentrifuge tube. The beads were then washed three times with 2-ml RIPA buffer and once with 1× PBS with immobilization and solution removal performed on a magnetic stand.

Samples were prepared for MS as follows. After the final wash, the beads were resuspended in 100 µl of 50 mM ammonium bicarbonate (Thermo Fisher Scientific), and the proteins on the beads were reduced with 10 mM DTT for 30 min at room temperature and alkylated with 55 mM iodoacetamide (Sigma-Aldrich) for 30 min in the dark. Protein digestion was performed with sequencing grade modified trypsin (Promega) at 1/50 protease/protein (wt/wt) at 37°C overnight. After trypsin digestion, the beads were washed twice with 100 µl of 80% acetonitrile (Thermo Fisher Scientific) in 1% formic acid (Thermo Fisher Scientific), and the supernatants were collected. Samples were dried in Speed-Vac (Thermo Fisher Scientific) and desalted and concentrated on a C18 Tip (Thermo Fisher Scientific).

On-bead digested samples were analyzed on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) coupled to an Easy-nLC 1200 system (Thermo Fisher Scientific) through a nanoelectrospray ion source. Peptides were separated on a self-made C18 analytical column (100 µm internal diameter × 20 cm length) packed with 2.7 µm Cortecs particles. After equilibration with 3 µl 5% acetonitrile and 0.1% formic acid mixture, the peptides were separated by a 120-min linear gradient from 6 to 42% acetonitrile with 0.1% formic acid at 400 nl/min. Liquid chromatography (Optima LC/MS; Thermo Fisher Scientific) mobile phase solvents and sample dilutions were all made in 0.1% formic acid diluted in water (buffer A) and 0.1% formic acid in 80% acetonitrile (buffer B). Data acquisition was performed using the instrument-supplied Xcalibur (version 4.1) software. Survey scans covering the mass range of 350–1,800 were performed in the Orbitrap by scanning from a mass/charge ratio (m/z) of 300–1800 with a resolution of 120,000 (at m/z 200), an S-Lens RF Level of 30%, a maximum injection time of 50 ms, and an automatic gain control target value of 4 × 10⁵. For MS2 scan triggering, monoisotopic precursor selectifon was enabled, charge state filtering was limited to 2–7, an intensity threshold of 2 × 10⁴ was used, and dynamic exclusion of previously selected masses was enabled for 45 s with a tolerance of 10 ppm. MS2 scans were acquired in the Orbitrap mode with a maximum injection time of 35 ms, quadrupole isolation, an isolation window of 1.6 m/z, higher-energy collisional dissociation of 30%, and an automatic gain control target value of 5 × 10⁴.

MS/MS spectra were extracted from raw data files and converted into .mgf files using a Proteome Discoverer Software (version 2.1.0.62). These .mgf files were then independently searched against human database using an in-house Mascot server (version 2.6; Matrix Science). Mass tolerances were ±10 ppm for MS peaks, and ±25 ppm for MS/MS fragment ions. Trypsin specificity was used, allowing for one missed cleavage. Met oxidation, protein amino-terminal acetylation, amino-terminal biotinylation, lysine biotinylation, and peptide amino-terminal pyroglutamic acid formation were allowed as variable modifications, while carbamidomethyl of Cys was set as a fixed modification. Scaffold (version 4.8; Proteome Software) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at >95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at >99.0% probability and contained at least two identified unique peptides.

To estimate relative protein levels, distributed normalized spectral abundance factors (dNSAFs) were calculated for each nonredundant protein, as described previously (Zhang et al., 2010). Average dNSAFs were calculated for each protein using replicates with nonzero dNSAF values. Enrichment of proteins in streptavidin affinity purifications from KIF1C-BioID-3xFLAG–tagged stable cell line relative to a control BioID stable cell line was calculated for all replicates as the ratio of average dNSAF (ratio = average dNSAF_KIF1C-BioID:average dNSAF_BioID). The volcano plot (Fig. 1 D) was generated by plotting the log₂(fold enrichment) against the −log₁₀(P value), where the P value (two-tailed Student’s t test) was computed by comparing the replicate dNSAF values of KIF1C-BioID to the BioID control. Potential KIF1C interactions were included as significant if they were not present in the control samples or were more than threefold enriched in the KIF1C-BioID-3xFLAG dataset and had P values <0.05.