SILAC Proteomics of Cobl in HEK293T

For SILAC, HEK293T cells stably expressing either 3xFLAG-Empty Vector or 3xFLAG-Cobl-COBL were grown in MEM with 10% dialyzed FBS, 2 μg/ml puromycin (Invitrogen) and either [¹²C]arginine and lysine or [¹³C]arginine and lysine, respectively, for 3 wk to allow uniform labeling of all proteins. FLAG immunoprecipitations were performed as described, with modifications for mass spectrometry (Smolka et al., 2007 (link); Viswanatha et al., 2012 (link)). Briefly, after immunoprecipitation, protein bound to FLAG resin was eluted in 50 mM Tris (pH 8.0) and 1% SDS and then precipitated with 50% ethanol, 49.9% acetone, and 0.1% acetic acid. Protein samples were then mixed, trypsin digested (Promega, Madison, WI) overnight at 37°C, and desalted in a C18 column (Waters, Milford, MA). The tryptic peptides were dehydrated in a speed vacuum and dissolved in 80% acetonitrile and 1% formic acid for fractionation by hydrophilic interaction chromatography. The resulting fractions were dried, dissolved in 0.1% trifluoroacetic acid, and injected into a mass spectrometer (Qexactive LC-MS/MS; Thermo Fisher Scientific). The data were analyzed using Proteome Discoverer (Thermo Fisher Scientific).