Qualitative proteomic analysis of TUBE-enriched ubiquitylome

SHSY5Y cells were seeded in 150 mm tissue culture plates and grown in DMEM/F12 (1:1) supplemented with 10% FBS and penicillin/streptomycin (1X). When cells were 80% confluent, a treatment time course was initiated. Cells were treated for 6, 12, 18, and 24 hours with 20 µM NAB2 in DMSO (and a DMSO control at 24 hour timepoint). Following treatment, cells were harvested and resuspended in TUBE lysis buffer (50mM Tris-HCl, pH 7.5, 0.15M NaCl, 1mM EDTA, 1% NP-40, 10% glycerol, published by LifeSensors, Inc.) with 1X protease inhibitor cocktail (Bimake) and 50 µM PR-619 (non-specific DUB inhibitor, Sigma). Cells were then sonicated (Fisher Scientific Model 120 Sonic Dismembrator; 4 pulses, 5 seconds per pulse, 30% amplitude) and cell debris was collected by centrifugation (14,000 rpm, 4 °C, 10 minutes). Lysate concentration was determined by Bradford assay. Magnetic TUBE 1 beads (pan-selective) were equilibrated by washing with TBS-T and lysate was added to TUBE beads to a final ratio of 100 µL bead slurry to 1 mg total protein. An additional sample was prepared with magnetic control beads (LifeSensors) and DMSO-treated lysate. Lysate was incubated with TUBE beads or control beads for 2 hours at 4 °C with end-over-end rotation. Following incubation, beads were collected and supernatant was removed. Beads were washed with TBST and bound protein was eluted for proteomic analysis with 50 mM TEAB buffer containing 5% SDS. Subsequently, a BCA assay was performed on the samples. The control pulldown had ~0.3 µg/µl and the other samples were 0.55, 0.52, 0.59, 0.5 and 0.53 µg/µl (for DMSO-treated, 6hr, 12hr, 18hr, and 24 hr timepoints, respectively). 25 µL of each sample was reduced and alkylated, followed by clean up and digestion with trypsin using an S-Trap (Protifi). After lyophilization, tryptic digests were resuspended in 12 µL, and 1 µL of each sample was analyzed by LC-MS/MS (see below) followed by database searching using Mascot. Search results were annotated at a 1% peptide/protein FDR in Scaffold.