Treated trypsin

BioID and mass spectrometry were conducted according to the protocol from Coyaud et al. (Mol. Cell. Proteomics 2015) [83 (link)]. Cells were grown in five 15 cm cell culture dishes until 70% confluence. Cells were incubated for 24 h in complete media supplemented with 1 µg/mL tetracycline (BioShop, Burlington, ON, Canada) and 50 µM biotin (BioShop, Burlington, ON, Canada) 8 h post initial induction. Cells were lysed, sonicated twice for 10 s at 35% amplitude (Sonic Dismembrator 500; Fisher Scientific, Waltham, MA, USA) and centrifuged at 16,000 rpm (35,000× g) for 30 min at 4 °C. Supernatants were passed through a Micro Bio-Spin Chromatography column (Bio-Rad 732-6204, Hercules, CA, USA) and incubated with 30µL of high-performance streptavidin-packed beads (GE Healthcare, Chicago, IL, USA) for 3 h at 4 °C on an end-over-end rotator. Beads were collected (2000 rpm, 2 min) and washed six times with 50 mm ammonium bicarbonate (pH8.3). Beads were then treated with L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Promega, Madison, WI, USA) for 16 h at 37 °C on an end-over-end rotator. Another 1 µL of TPCK-trypsin was added and incubated in a water bath at 37 °C for 2 h. Supernatants were lyophilized and stored at 4 °C for downstream mass spectrometry analysis.