Protein Extraction and Digestion Protocol

Spinal cord neuron-neuroblastoma (NSC-34) (CED-CLU140, Biozol, Eching, Germany), mouse embryonic fibroblasts (MEFs) (American Type Culture Collection, Manassas, VA), and mouse hepatoma (liver cancer, Hepa 1–6) (CRL-1830, American Type Culture Collection) cell lines were cultured and proteins prepared as previously described (28 (link)). Briefly, the cells were lysed in lysis buffer (4% SDS, 10 mm Hepes, pH 8.0) during sonication for 15 min (level 5, Bioruptor; Diagenode, Seraing (Ougrée) - Belgium). Cell lysis was followed by reduction of disulfide bonds with 10 mm DTT for 30 min and subsequent alkylation with 55 mm IAA for 45 min. To remove the detergent, cold acetone (−20 °C) was added to 100 μg of proteins to a final concentration of 80% v/v, and proteins were precipitated for at least 2 h at −20 °C. The suspension was centrifuged for 15 min (4 °C, 16,000 × g) and the precipitate was washed with 80% acetone (−20 °C) prior to re-suspension in 50 μl of 6 m urea/2 m thiourea, 10 mm Hepes, pH 8.0. An initial digestion step (3 h) was carried out after the addition of 1 μg of LysC, followed by dilution with four volumes of 50 mm ammonium bicarbonate and the final digestion with 1 μg of trypsin overnight at room temperature. The resulting peptide mixtures were desalted on SDB-RPS StageTips (29 (link)) and subjected to single shot LC-MS/MS analysis.