TRAF6 substrate profiling by Ub-BOD

Version 1

After respective treatment, cells were washed 2 times with ice-cold DPBS, scraped in PBS and pellets either processed immediately or stored at -80°C. Cells were lysed in RIPA buffer for 30 min on ice. After clearance by centrifugation at 20,000 × g for 10 min at 4 °C, protein concentrations were adjusted using BCA assay. Cleared and adjusted supernatants were incubated overnight on an overhead rotator with preequilibrated streptavidin agarose (Sigma). Next day, beads were washed 2 times with RIPA buffer, 4 times with freshly prepared 3 M Urea buffer (Urea in 50 mM ammonium bicarbonate) and suspended in a defined volume of 3 M Urea buffer. Samples were reduced with 5 mM TCEP (Sigma) at 55 °C for 30 min, alkylated with 10 mM IAA (Sigma) at room temperature for 20 min and quenched with 20 mM DTT (Sigma). Samples were washed two times with freshly prepared 2 M Urea buffer (Urea in 50 mM ammonium bicarbonate), suspended in 50 μl of 2 M Urea buffer and digested with 1 μg trypsin per sample at 37 °C overnight. Peptides were collected by pooling the supernatant with two 50 μl 2 M Urea buffer washes, immediately acidified with 1% trifluoroacetic acid and concentrated by vacuum centrifugation. Digested peptides were desalted on custom-made C18 stage tips and reconstituted in 0.1% formic acid.