Surface biotinylation assay and proteomics

All buffers were filter sterilized prior to use. Each sample required 24 million cells that were pelted and washed twice with PBS. 10 mM of freshly prepared EZ-Link™ Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific, A39258) in PBS and added to the cell mixture at 80 µl of 10 mM NHS-SS-Biotin per ml of sample volume. Samples were incubated by rocking at room temperature for 30 min. After incubation, cells were pelleted at 300 g-force for 3 min, excess biotin discarded and then washed twice with ice-cold TBS.

Proteins were collected from cells via lysing with dissolved Pierce™ Protease Inhibitor Mini Tablets, EDTA-free (Thermo Fisher Scientific, A32955) in RIPA buffer (Thermo Fisher Scientific, 89901). Samples were sonicated for 15 s at 15% amplitude, followed by vortexing for 10 s every 10 min for 30 min. This was followed by a 5 min centrifugation at 15,000 g-force and supernatant.

Enrichment of biotinylated proteins proceeded via an overnight incubation at 4°C on NeutrAvidin resin (Thermo Fisher Scientific, 53150) at a ratio of 10 µl of beads per 100 µg protein. After incubation, samples were centrifuged at 700 g-force for 2 min at 4°C. Pellets then underwent a series of wash steps: twice with lysis buffer, thrice with ultrapure H₂O and finally, twice with 50 mM ammonium bicarbonate (ABC), pH 8.0. A portion of clarified proteins were assessed for biotinylation efficiency via western blotting with a streptavidin secondary antibody (Li-Cor, 32230). Biotinylated proteins were eluted from resin using 10 mM dithiothreitol (Thermo Fisher Scientific, R0861) dissolved in 50 mM ABC by end-over-end rotation at room temperature for 30 min. Sample was centrifuged at 700 g-force for 2 min and supernatant collected.

Sample preparation for mass spectrometry required the addition of 25 µl of freshly prepared 55 mM iodoacetamide (dissolved in 50 mM ABC) with a requisite 30 min incubation at room temperature in the dark. After the time interval, we added ice-cold acetone to mix overnight at −20°C. Samples were then centrifuged at 15,000 g-force for 10 min, and then decanted for 30 min at room temperature to precipitate proteins.

Precipitated proteins were resuspended in 25 mM ammonium bicarbonate, digested overnight using 1 µg of trypsin (Promega, V5113) at 37°C. Samples were acidified with 10% v/v of trifluoroacetic acid, desalted using C18-stage tips, and dried. For MS analysis, peptides were resuspended in 1% formic acid and separated on an Easy nLC 1000 UHPLC equipped with a 15 cm nanoLC column. Using a flow rate of 300 nl/min, the linear gradient was 5% to 35% over B for 90 min, 35% to 95% over B for 5 min, and 95% hold over B for 15 min (solvent A: 0.1% formic acid (FA) in water, solvent B: 0.1% FA in ACN). The table indicates ley mass spectrometer parameters.

Peptide identities and relative abundances were determined using Proteome Discoverer 2.4. Ion chromatograms were extracted using Xcalibur Qual Browser for each peptide of interest with a mass tolerance of 0.5 Da. We thank Dr. Steve Eyles (University of Massachusetts Amherst, MA, USA RRID: SCR-019063) for assistance with high-resolution MS acquired on an Orbitrap Fusion mass spectrometer (National Institutes of Health grant: 1S10OD010645-01A1).