Peroxisomes are primarily metabolic organelles with important functions in lipid metabolism, such as fatty acid oxidation and ether phospholipid synthesis (e.g., plasmalogens). Certain viruses, such as human cytomegalovirus (HCMV), hijack organelle functions to facilitate their replication and spread. However, the role of peroxisomes in herpesvirus replication remains elusive. Following a discovery that peroxisome proteins are upregulated upon HCMV infection, we quantified the production of plasmalogens, lipids that require peroxisome functions. In agreement with the increase in peroxisome protein abundance, plasmalogen production was increased by HCMV infection.

Three mass spectrometry-based quantitative analyses of plasmalogens were performed. First, primary human fibroblasts grown in tissue culture were either harvested uninfected, or infected and harvested at 120 hours post infection for plasmalogen quantification. Second, plasmalogens were quantified from two peroxisome knockout cell lines and a non-targeting knockout control in uninfected conditions. Lastly, plasmalogens were quantified from two peroxisome knockout cell lines and the control in infected conditions at 120 hours post infection. All analyses were performed in biological triplicate.

Phospholipids were isolated for MS analysis using the Bligh-Dyer method. A total of 1x10^6 cells were incubated in serum-free DMEM for 24 hours prior to processing. Cells were recovered by scrapping in 0.8ml of ice-cold 1:1 0.1N HCl:MetOH (v/v) and transferred to a microcentrifuge tube. A total of 200ng odd-carbon chain internal standard 17:0/17:0 PE (Avanti Polar Lipids, Inc.; No. 830756) and 0.4ml ice-cold chloroform were added. The sample was vortexed and centrifuged (5 min, 18,000xg). The organic phase was collected, solvent evaporated (Speedvac SC110A, ThermoSavant), and dissolved in 50μl methanol prior to injection of 20μl for LC-MS analysis. A Shimadzu UFLC system coupled to a Thermo LTQ XL mass spectrometer was used for LC-MS analysis in positive-ion mode. Phospholipids were separated in a 60-minute stepped linear reverse-phase gradient (solvent B set to 0% 5min, 30% 15 min, 50% 60min) using an ACE C18 column (3μm, 75mm x 1mm) at 45°C. Solvent composition for buffer A (2:2:1 acetonitrile:methanol:water (v/v/v) supplemented with 0.1% formic acid and 0.028% ammonia) and buffer B (isopropanol supplemented with 0.1% formic acid and 0.028% ammonia). Scan range was set to 680-820 m/z with a 60,000 resolution for MS1 in the FT and 200-800 m/z for MS2 in the IT. Instrument was set to perform a full MS scan followed by isolation and collision induced dissociation (CID) for MS2 acquisition from an inclusion list containing m/z for phosphatidylethanolamine (PE) plasmalogen precursor [M+H]+ and for 17:0/17:0 PE internal standard (m/z = 720.5). Isolation window set to 1.9 m/z, normalized collision energy (NCE) to 32, acquisition Q of 0.250, and activation time of 30ms.