Aqua 5 m

HEK293T cells were transfected with HA-DCAF16 plasmid by PEI transfection reagent for 24 h and treated with DMSO or 10 µM KB02-PEG0-SLF for 2 h. Cells were collected and lysed in 1% NP-40 lysis buffer with complete protease inhibitor cocktail. HA immunoprecipitation (40 µL slurry anti-HA agarose, 4 °C, 2 h) was performed with 10 mg of total protein lysates to purify HA-DCAF16. After washing the HA resin three times with IP washing buffer and once with PBS, HA-DCAF16 protein was eluted by heating at 65 ºC for 10 min with 8 M urea in PBS, then reduced with 12.5 mM DTT at 65 ºC for 15 min and alkylated with 25 mM iodoacetamide at 37 ºC for 30 min. The protein solution was diluted with PBS to 2 M urea and digested with 2 µg trypsin at 37 ºC for 6 h. Tryptic peptides were acidified with 5% formic acid and loaded onto a silica capillary column (250 µm) packed with 3 cm of C18 resin (Aqua 5 µm, Phenomenex). Peptides were analyzed on LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific) coupled with a Thermo UltiMate 3000 UHPLC system. Peptides were separated on a capillary column packed with 10 cm of C18 resin (Aqua 5 µm, Phenomenex) and a 5 µm tip. MS parameters were set as previously described⁵¹ . The raw data was acquired in Xcalibur operation software.

TEV digested samples were pressure-loaded onto a 250 µm (inner diameter) fused silica capillary columns packed with C18 resin (Aqua 5 µm, Phenomenex) and analyzed by multidimensional liquid chromatography tandem mass spectrometry (MudPIT) using an LTQ-Velos Orbitrap mass spectrometer (Thermo Scientific) coupled to an Agilent 1200-series quaternary pump. The peptides were eluted onto a biphasic column with a 5 µm tip (100 µm fused silica, packed with 10 cm of C18 resin and 4 cm of bulk strong cation exchange resin (SCX, Phenomenex) in a 5-step MudPIT experiment, using 0%, 30%, 60%, 90%, and 100% salt ‘bumps’ of 500 mM aqueous ammonium acetate and 5%→100% gradient of buffer B in buffer A (buffer A: 95% water, 5% acetonitrile, 0.1% formic acid; buffer B: 5% water, 95% acetonitrile, 0.1% formic acid) as previously described (Weerapana et al., 2007). Acquired data was collected in a data-dependent acquisition mode with dynamic exclusion enabled (20 s, repeat count of 2). One full MS (MS1) scan (400–1800 m/z) was followed by 30 MS2 scans (ITMS) of the nth most abundant ions.