Cell lysis and digestion
Cells were lysed using 1% IGEPAL (Sigma) in PBS (Invitrogen) for 20 minutes at room temperature (RT) to inactivate the virus. These specific lysis conditions were used as this was the approved virus inactivation protocol. Proteins contained in the cell lysate were then immediately precipitated using 90% methanol (v/v) (Sigma) by centrifugation at 20,000x g for 10 minutes. The protein pellets were frozen at −80°C. Precipitated proteins were resuspended in lysis buffer (8 M urea, 100 mM ammonium bicarbonate (ABC), 150 mM NaCl, protease inhibitor (mini-cOmplete, Roche) and phosphatase inhibitors (phosSTOP, Roche). Tris-(2-carboxyethyl)phosphine (TCEP) was added to a final concentration of 4 mM. DNA was sheared via probe sonication, on ice, at 20% amplitude for 20 s., followed by 10 s of rest. This process was performed a total of three times. Following sonication, protein concentration was determined using Bradford assay. Iodoacetamide (IAA) was added to each sample to a final concentration of 10 mM, and samples were incubated in the dark at room temperature (RT) for 30 minutes. Excess IAA was quenched by the addition of dithiothreitol (DTT) to 10 mM, followed by incubation in the dark at RT for 30 minutes. Samples were then diluted with 0.1 M ABC (pH = 8.0) to a final urea concentration of 2 M. Trypsin (Promega) was added at a 1:100 (enzyme:protein w:w) ratio and digested overnight at 37°C with rotation. Following digestion, 10% trifluoroacetic acid (TFA) was added to each sample to a final pH ∼2. Samples were desalted under vacuum using Sep Pak tC18 cartridges (Waters). Each cartridge was activated with 1 mL 80% acetonitrile (ACN)/0.1% TFA, then equilibrated with 3 × 1 mL of 0.1% TFA. Following sample loading, cartridges were washed with 4 × 1 mL of 0.1% TFA, and samples were eluted with 4 × 0.5 mL 50% ACN/0.25% formic acid (FA). 20 μg of each sample was kept for protein abundance measurements, and the remainder was used for phosphopeptide enrichment. Samples were dried by vacuum centrifugation.