At various time points following addition of labeled DMEM, metabolites were harvested as previously described.20 (link) After drying the samples, the metabolites extracted from the infected plates were dissolved in 600 µL of HPLC-grade water, while metabolites from mock-treated plates were dissolved in 300 µL. This 2-fold difference in volume accounts for the ~2-fold increase in volume of the fibroblasts during human cytomegalovirus infection. Volumes of 10 µL of each metabolite extract were analyzed via reversephase ion-pairing chromatography coupled to a stand-alone orbitrap mass spectrometer. The mass spectrometer scan rate was set to 1 Hz and resolving power to 100 000, scanning m/z 85–1000 in the negative ion mode. All other parameters are as in Lu et al.21 (link) The LC gradient was 0 min, 0% B; 2.5 min, 0% B; 5 min, 20% B; 7.5 min, 20% B; 13 min, 55% B; 15.5 min, 95% B; 18.5 min, 95% B; 19 min, 0% B; 25 min, 0% B. Solvent A is 97:3 water–methanol with 10 mM tributylamine and 15 mM acetic acid; solvent B is methanol. The flow rate was 200 µL/min on a Synergy Hydro-RP column (100 mm × 2 mm, 2.5 µm particle size, Phenomenex, Torrance, CA).