Stable isotope labeling of amino acids in cell culture (SILAC) during viral infection

To assess the newly synthesized proteins upon viral infection, we employed SILAC labeling, comparing virally infected cells with that of an uninfected population. To do so, iBMDMs were seeded in tissue-culture treated 6-well plates (1 million cells per well) and primed for at least 6 h with IFNγ. Infection with mAdV2 or mAdV3 was performed by adding 750μl viral suspension in iBMDM media containing R⁺¹⁰ (U-13C6 99%,15N2 99%, Cambridge Isotope Laboratories) and K⁺⁸ (U-13C6 99%; U-15N4 99%, Cambridge Isotope Laboratories) (SILAC heavy media), using iBMDM media containing R⁺⁶ (13C6, 99%, CIL) and K⁺⁴ (4,4,5,5-D4, 96-98%, CIL) (SILAC intermediate media) as uninfected control. At 16hpi, media was removed, cells were washed twice in PBS and subsequently lysed in 500 μl 100 mM Tris pH7.5, containing 4% SDS (FASP-buffer), supplemented with 10 mM DTT (Merck). Lysates were sonicated to shear DNA, boiled at 95 °C for 5 minutes and cleared by centrifugation at full speed for 10 min. Cleared lysates were further processed at the Protein Analysis Facility of the University of Lausanne.

After determination of protein concentration (tryptophan fluorescence method⁹⁵), H and I samples were mixed at an equimolar ratio (total: 100μg) and digested (SP3 method⁹⁶ using magnetic Sera-Mag Speedbeads (Cytiva 45152105050250, 50 mg/ml). To alkylate, proteins were treated with 32 mM iodoacetamide (final concentration) for 45 min at RT in the dark. Precipitation was done on beads (10:1 (w:w) ratio beads:material) using ethanol (final concentration: 60%), and after 3 washes with 80% ethanol, beads were digested in 100 mM ammonium bicarbonate containing 1μg trypsin (Promega #V5073), final volume 50 μl, for 2 h at 37 °C. The same amount of trypsin was added for an additional 1 h of digest. Supernatants were recovered and mixed with two sample volumes of isopropanol containing 1% TFA. Samples were desalted on a strong cation exchange (SCX) plate (Oasis MCX; Waters Corp., Milford, MA) by centrifugation, washed with isopropanol containing 1%TFA, eluted in 200μl 80% MeCN, 19% water, 1% (v/v) ammonia, and dried by centrifugal evaporation.