Trypsin Digestion and Dimethyl Labeling

The proteins were digested with trypsin as previously described [37 (link)]. In detail, the protein sample was diluted after adding 100 mM NH₄CO₃ to a urea concentration of less than 2 M. Then, trypsin (Promega, Madison, CT, USA) was added at 1:50 trypsin-to-protein mass ratio for the first digestion at 37 °C overnight and a 1:100 trypsin-to-protein mass ratio for a second four-hour digestion. After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex, Torrance, CA, USA) and vacuum-dried. Peptide was reconstituted in 0.1 M NaAc (pH = 5.99). The samples are differentially isotope labeled in parallel in different tubes by adding four µL CH₂O or CD₂O to the samples to be labeled with light (control Cell) and heavy (viral infected cell) dimethyl, respectively. The reactions were mixed briefly and added four µL of 0.6 M NaBH₃CN, incubated in a fume hood for one hour at room temperature. Finally, the labeling reactions were quenched by adding 16 µL of 1% ammonia solution, then adding formic acid for desalted and dried by vacuum centrifugation. Then the tryptic peptides were subjected to lysine acetylated peptide enrichment using a previously described method [66 (link)]. The resulting peptides were cleaned with C18 ZipTips (Merck Millipore) according to the manufacturer’s instructions, followed by LC–MS/MS analysis.