Trypsin lys c protease mix

Protein extracts containing VHL-interacting proteins were resuspended in 100 μL of 75 mM ammonium bicarbonate buffer pH 8.0. Proteins were reduced with 10 mM DTT for 20 min and alkylated in the dark with 50 mM iodoacetamide. Incubations were performed at room temperature on a tube rotator to ensure homogeneous suspension of the beads. Two micrograms of a Trypsin/Lys-C protease mix (Promega Corporation, USA) were added to samples and incubated overnight at 37°C on a tube rotator. Samples were supplemented with 2 μg of SOLu-Trypsin (Millipore-Sigma, USA) and incubated for an additional 3 hrs at 37°C to promote the completeness of the digestion process. The digested products were acidified with trifluoroacetic acid (TFA) and the peptides were isolated and washed on C18 tips (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Finally, the purified peptides were dried by vacuum centrifugation and stored at −80°C.

Eluted protein samples (150 μl) were reduced by adding 4 μl of 500 mM TCEP [Tris(2-carboxyethyl)phosphine]–100 mM TEAB (triethylammonium bicarbonate) for 1 h at 55°C. A 4-μl volume of 625 mM iodoacetamide was added to each sample followed by incubation for 30 min at room temperature in the dark. Proteins were then precipitated using the methanol-chloroform-water method (67 (link)). Protein samples were resolubilized in 100 μl of 100 mM TEAB and sonicated for 10 min. The protein content was measured with a Merck Millipore Direct Detect infrared spectrometer. The samples were treated with trypsin/Lys-C protease mix (Promega, catalog no. fV5072) at a protease/protein ratio of 1:25 for 16 h at 37°C. The reaction was stopped with 10 μl of 10% (vol/vol) formic acid, and the reaction mixture was subjected to evaporation using a SpeedVac (Thermo Fisher Scientific). Peptides were resolubilized in 25 μl 0.05% (vol/vol) trifluoroacetic acid (TFA)–2% (vol/vol) acetonitrile (ACN)–water and sonicated for 15 min in a water bath before transfer to a 10-kDa-molecular-weight cutoff filter. After 15 min of centrifugation at 14,000 × g (4°C), the samples were transferred to high-performance liquid chromatography (HPLC) vials and stored at –20°C.