Shotgun proteomics

LCL cells were cultured in lysine- and arginine-free DMEM (GIBCO) supplemented with 2 mM glutamine, 10% dialyzed FBS and antibiotics as well as with 146 mg/ml light (K0, Sigma) or heavy lysine (K8, Cambridge Isotope Laboratories) and 84 mg/ml light (R0, Sigma) or heavy (R10, CambridgeIsotope Laboratories) arginine, respectively. Cell pellets were lysed in 8 M Urea in 50 mM Tris-HCl pH 8.0. Protein concentrations were determined by a BCA assay and protein amounts were adjusted to equal concentrations. Cell lysates were reduced by 1 mM DTT, alkylated by 5 mM iodoacetamide, and digested by Lys C for 4 h. The urea solution was diluted to 1 M urea before tryptic digestion overnight. Resulting peptides were purified and fractionated as described previously (Hu et al., 2019). Phosphopeptide enrichment was performed on an Automated Liquid Handling Platform (AssayMap Bravo, Agilent) (Post et al., 2017) using Fe (III)-NTA cartridges (5 µL).

For whole cell proteome analysis: Cell pellets were resuspended in urea buffer (9 M Urea, 50 mM Tris pH 8, 150 mM NaCl, 1x protease inhibitors (Roche), 50 µM PR-619) and sonicated. The sample was cleared by centrifugation at 2500 g and protein amount was adapted after a BCA assay. DTT (5 mM final) was added and incubated for 25 min at 56°C for protein reduction. Samples were then incubated for 30 min at room temperature in the dark with IAA (14 mM final) for protein alkylation. The reaction was quenched by addition of DTT (5 mM final). The protein mixtures were diluted 1:5 with 1 M Tris-HCl, pH 8.2 to lower the urea concentration. Protein was digested with LysC (FUJIFILM, 2 µl/100 µg protein) for 3 h followed by trypsin (0.5 µg/100 µg protein) at 37°C overnight. Digestion was stopped by the addition of 10% TFA. Peptide samples were fractioned by C18-SCX custom-made stage tips as described elsewhere (Rappsilber et al, 2007). Briefly, the sample was loaded on a pre-conditioned stage tip containing 2x SCX disks and 2x C18 disks. Stepwise elution was performed with reversed phase-ion exchange buffers (ReX Buffer: 0.5% AcOH, 20% ACN) with increasing NH4AcO concentrations (20 mM to 500 mM). The collected fractions were desalted on custom made C18 stage tips before MS analysis. In general, 4 biological replicates were used per cell line, except for UBQLN2-P497S LCLs and UBQLN2-T487I HeLa, where one replicate was lost during processing. Eluted peptides were loaded onto custom-made 75 mmx 15 cm fused silica capillaries filled with C18AQ resin ((Reprosil Pur 120, 1.9 µm, Dr. Maisch HPLC) using an Easy-nLC1200 liquid chromatography. Peptide mixtures were separated using an ACN gradient (5% to 95%) in 0.5% acetic acid on a Q Exactive HF mass spectrometer (Thermo Scientific). For APEX2-experiemnts a 35 min long gradient was used, while a 75 min long gradient was used for whole proteomics. For IP-MS: PBS-washed cell pellets were lysed in MCLB buffer (50mM Tris pH 7.4, 150mM NaCl, 0.5% NP40, 1x protease inhibitors (Roche)). Lysates were cleared by centrifugation (14000 rpm, 10 min, 4°C), concentrations between samples were adapted and then samples were filtered through a 45 µm spin filter (Millipore). Lysates were immunoprecipitated overnight at 4°C with pre-equilibrated anti-HA-agarose (Sigma). Agarose beads were washed 5x with ice cold MCLB followed by 5 washing steps with PBS. Afterwards, proteins were eluted with HA-Peptide (Sigma-Aldrich). TCA was added to the samples for a final concentration of 20% to precipitate proteins. For TCA removal, precipitated proteins were washed 3x with ice cold acetone. Precipitated proteins were solved in ammonium bicarbonate (ABC) with 10% acetonitrile and trypsin was added. After 4 h at 37°C the tryptic digest was stopped, and samples were desalted on custom made C18 stage tips before MS analysis (see above).