SILAC-Based Protein Quantification and Digestion

The protein concentration of the SILAC cell lysates was determined using the bicin chonicic acid assay (Pierce). Digestion of the proteins was performed using the Filter-Aided Sample Preparation (FASP) method [108 (link)], for which equal amounts (900 μg) of mock- and virus-infected cell lysates were mixed and DTT was added to a final concentration of 50 mM, followed by a 5-min incubation at 70°C. Samples were loaded on two 15-ml 30 kDa Microcon filter devices (Millipore), which were washed twice with 8 M urea 0.1 M Tris pH 8.5, while cysteines were alkylated with 50 mM iodoacetamide in the same buffer. Samples were washed 3 times with 8 M urea, 0.1 M Tris pH 8. Proteins were digested overnight at room temperature using 20 ug endoLysC (Wako Pure Chemical Industries) in the same buffer per filter device. The sample was diluted fourfold with 50 mM ammonium bicarbonate pH 8.4 containing 20 ug trypsin (Worthington Chemical Corporation), and digested for 4 h at room temperature. Peptides were collected by centrifugation, acidified to a final percentage of 1% TFA, and desalted using solid phase extraction. Peptides were eluted in 20/80/0.1 (v/v/v) of milliQ/acetonitrile (ACN) (Actu-All Chemicals)/trifluoric acid (TFA) (Sigma-Aldrich).