Shotgun proteomics

Immunoprecipitation: Cells grown in 2-4x15 cm cell culture plates per sample were harvested by scraping on ice and stored at -80. Lysis was performed for 30 min at 4°C with MCLB buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 0.5% NP40, 1x PhosStop,1x protease inhibitor) or Glycerol buffer (20 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 0.5% TritonX, 1x PhosStop, inhibitor, 1x protease inhibitor). Samples were cleared from debris by centrifugation (20.000 g for 10 min at 4°C) and Ultrafree®-CL spin-filter tubes. Protein concentrations of lysates were adjusted following determination by BCA and samples incubated overnight with pre-equilibrated anti-HA-agarose or anti-Flag M2 affinity gel using an overhead shaker at 4°C. Subsequently, agarose beads were washed five times with the respective buffer and eluted by boiling in SDS sample buffer (200 mMTris-HCL, 6 % SDS, 20 % Glycerol, 300 mM DTT and Bromophenol Blue) (5 mins at 95°C) or washed 5 more times with DPBS prior to elution with HA peptide. Eluted immune complexes were precipitated with TCA (final concentration 20%) and washed with ice cold acetone. Samples were resuspended in 50 mM ammonium bicarbonate buffer containing 10% acetonitrile and trypsinized for 4 h at 37°C. Proximity biotinylation: Cells were grown in the presence of 500 µM biotin-phenol for 30 min at 37°C and pulsed with 1 mM H2O2 at RT. Biotinylation was stopped by washing three times with quencher solution (10 mM sodium azide, 10 mM sodium ascorbate, 5 mM 6-Hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid, DPBS). The third quenching step was performed for 15 min before washing three times with DPBS. Biotinylated samples were thawed on ice and lysed in qRIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium desoxycholate, 1% Triton-X, 10 mM sodium azide, 10 mM sodium ascorbate, 5 mM TROLOX, PhosStop and protease inhibitor) for 40 min at 4°C. Samples were cleared from debris (20.000g, 10 min, 4°C) and incubated with pre-equilibrated streptavidin agarose beads overnight at 4°C in an overhead shaker. Pulldowns were washed twice with RIPA buffer followed by four times washing with 3 M urea wash buffer (50 mM ABC buffer, 3 M urea). TCEP was added to a final concentration of 5 mM and incubated for 30 min at 55°C. Once cooled to RT samples were incubated with iodoacetamide (IAA) for 20 min at RT in the dark followed by addition of DTT (20 mM). Samples were then washed with 2 M urea wash buffer (50 mM ABC buffer, 2 M urea) prior to trypsinization overnight at 37°C. DiGly: Cells were cultured in lysine- and arginine-free DMEM supplemented with dialyzed FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin/streptomycin and light (K0) lysine and arginine. Heavy media was the same except the light lysine was replaced with K8-lysine. Cells were washed twice with ice-cold PBS and lysed in 5 ml denaturing lysis buffer (8M Urea, 50 mM Tris [pH 8], 50 mM NaCl, 1X PIC [protease inhibitor cocktail, EDTA-free], 50 µM DUB inhibitor PR-619). Samples were incubated on ice for 10 min and then sonicated with 3x 20 s pulses. Following removal of non-solubilized material (15,000xg/10 min), differentially labeled lysates were mixed at equal ratios based on total protein determined by BCA. Following reduction with 5 mM DTT and alkylation with 10 mM chloroacetamide, lysates were digested with 5 ng/μl lys-C for 1 h at room temperature. Subsequent digestion of peptides with trypsin was performed as described. Lyophilized peptides were resuspended in 1.5 ml IAP buffer (50 mM MOPS [pH 7.4], 10 mM Na2HPO4, 50 mM NaCl) and centrifuged to remove any insoluble material (2500xg/5 min). The supernatant was incubated with anti-diGly antibody (32 μg/IP) conjugated to protein A agarose beads for 1 h at 4°C. Unbound peptides were removed through 3x washing with IAP buffer and once with PBS. Bound material was eluted 4x with 50 µl 0.15% TFA and peptides were desalted using C18 stage-tip method. Each sample was immunoprecipitated sequentially two times and each IP was analyzed separately.