High performance streptavidin packed beads

BioID-MS was conducted as described previously (42) . NLN or ClpP complementary DNA (cDNA) was fused in-frame with a mutant Escherichia coli biotin-conjugating enzyme, BirA R118G (or BirA*) into a tetracycline-inducible pcDNA5 FLP recombinase target/ tetracycline operator (FRT/TO) expression vector, which was then transfected into Flp-In T-REx HEK293 Flp-In cells. Cells were lysed, sonicated twice for 10 s at 35% amplitude (Sonic Dismembrator 500; Fisher Scientific), and centrifuged at 35,000g for 30 min at 4°C. Supernatants were passed through a Micro Bio-Spin chromatography column (Bio-Rad 732-6204) and incubated with 30 l of high-performance streptavidin packed beads (GE Healthcare) for 3 hours 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 (pH 8.3). Beads were then treated with l-1-tosylamide- 2-phenylethyl chloromethyl ketone (TPCK)-trypsin (Promega) for 16 hours at 37°C on an end-over-end rotator. After 16 hours, another 1 l of TPCK-trypsin was added for 2 hours and incubated in a water bath at 37°C. Supernatants were lyophilized and stored at 4°C for downstream MS analysis. Two biological and two technical replicates were completed for each protease. NLN's interactors were compared to ClpP's interactors, which were normalized to each protease's respective BirA* spectral counts.