SILAC-based FLAG Immunoprecipitation and Mass Spectrometry

For SILAC, JEG-3 stable cell lines expressing 3xFLAG-E3KARP were grown for ∼3 wk in MEM (Thermo Fisher Scientific) containing dialyzed FBS (Invitrogen) and either C¹²-arginine and lysine or C¹³-arginine and lysine (Sigma-Aldrich), respectively. FLAG immunoprecipitations were as described, with slight modifications for mass spectrometry processing, as described previously (Smolka et al., 2007 (link); Viswanatha et al., 2012 (link)). Briefly, after immunoprecipitation, bound protein 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), and desalted in a C18 column (Waters, Milford, MA). The tryptic peptides were dehydrated in a speed vacuum and then resuspended in 1% acetic acid. Phosphopeptides were enriched by IMAC as previously described (Smolka et al., 2007 (link); Albuquerque et al., 2008 (link); Ohouo et al., 2013 (link)), reconstituted in 85 μl of solution containing 80% acetonitrile and 1% formic acid, and fractionated by hydrophilic interaction liquid chromatography. The resulting fractions were injected into a mass spectrometer (Qexactive LC-MS/MS; Thermo Fisher Scientific) and the data analyzed using Proteome Discoverer (Thermo Fisher Scientific).