Affinity purification coupled with mass spectrometry proteomics

For diGly proteomics: HeLa 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 (38 μg/mL) and arginine (66 μg/ml). Medium and heavy media were the same except the light lysine was replaced with K4 and K8-lysine, respectively. Medium and heavy labeled cells were treated for 1 h with 250 µM LLOMe while light labeled were treated for 1 h with vehicle alone (EtOH). Light and heavy cells were repeatedly washed with fresh SILAC medium and cultured without LLOMe for another 2 hrs. 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, 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 peptides were digested with trypsin over night. 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. The supernatant was incubated with anti-diGly antibody 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 three times and each IP was analyzed separately by mass spectrometry. Peptides samples were separated on a nanoflow HPLC system using a 226 min gradient of 5-33% acetonitrile containing 0.5% acetic acid on custom filled C18 reversed-phase columns and analyzed on a hybrid ion-trap Orbitrap mass spectrometer (Orbitrap Elite, Thermo Scientific) using data-dependent acquisition selecting the most intense peaks from each full MS scan acquired in the Orbitrap for subsequent MS/MS while excluding peptides with unassigned charge states or charge states below +3 from fragmentation. For proximity proteomics: HeLa cells stably expressing CNN2-APEX2 were grown in lysine- and arginine-free DMEM supplemented with FBS, L-Glutamine, Sodium pyruvate, heavy arginine (R10) (38 μg/ml) and lysine (K8) (66 μg/ml) or light arginine (R0) (38 μg/ml) and lysine (K0) (66 μg/ml), respectively. Heavy-labeled cells were treated with 250 μM LLOMe for 3 h at 37°C, while light-labelled cells were treated with vehicle alone (EtOH). Cells were incubated with 500 µM Biotin-Phenol during the last 30 min of LLOMe treatment and subsequently pulsed by addition of H2O2 for 1 min at room temperature. To stop the biotinylation reaction, they were washed 3x with quencher solution (10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox in DPBS) and 3x with PBS. All further steps were performed at 4°C unless indicated otherwise. After cell harvest with 0.25% Trypsin/EDTA, cells were counted and heavy- and light-labelled cells were mixed at a 1:1 ratio based on total cell numbers. After centrifugation, the resulting cell pellets were lysed in APEX-RIPA (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate supplemented with 10 mM sodium ascorbate, 1 mM sodium azide, 1 mM Trolox and protease inhibitors). Samples were sonicated 2x for 1 s, spun down at 10,000xg for 10 min before application to streptavidin agarose resin and incubation with overhead shaking overnight. Subsequently, samples were washed 3x in APEX-RIPA buffer and 3x in 3 M Urea buffer (in 50 mM ABC) followed by incubation with TCEP (5 mM final) for 30 min at 55°C with shaking. After alkylation with IAA (10 mM final) for 20 min at room temperature in the dark the reaction was quenched with DTT (20 mM final). Samples were washed 2x with 2 M Urea (in 50 mM ABC) before trypsin digestion overnight at 37°C (20 µg/ml final). The resin was spun down and supernatants containing digested peptides were collected. After washing the resin 2x with 2 M Urea and pooling all supernatants the samples were acidified with TFA (1% final). Digested peptides were desalted on custom-made C18 stage tips. Using an Easy-nLC1200 liquid chromatography, peptides were loaded onto custom filled C18 reversed-phase columns and separated using a gradient of 5%–33% acetonitrile in 0.5% acetic acid over 90 min and detected on an Q Exactive HF mass spectrometer (ThermoFisher Scientific). Dynamic exclusion was enabled for 30 s and singly charged species or species for which a charge could not be assigned were rejected.