Targeted Quantification of the Peroxisome Proteome during Human Cytomegalovirus Infection
Infection-Induced Peroxisome Biogenesis Is a Metabolic Strategy for Herpesvirus Replication, Jean Beltran PM, Cook KC, Hashimoto Y, Galitzine C, Murray LA, Vitek O, Cristea IM
- Organism: Human, Human Cytomegalovirus
- Instrument: Q-Exactive HF
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. Therefore, we used targeted mass spectrometry to quantify 60 peroxisome proteins through the HCMV infection cycle.
A parallel reaction monitoring (PRM) method for quantification of peroxisome proteins was designed and used to analyze uninfected and infected cells. A total of 60 proteins, containing 160 peptides, were targeted for quantification of peroxisome proteins. Three viral proteins were used to monitor the progression of infection, histone peptides were used for abundance normalization, and tubulin was used as a control for a protein that does not change in abundance during infection. Primary human fibroblasts were grown in tissue culture and either harvested uninfected or infected and harvested at 6, 24, 48, 72, and 120 hours post infection for LC-MS analysis. This analysis was performed in biological triplicate.
Whole cells were lysed (4% SDS, 0.1M NaCl, 50mM TrisHcl, 0.5mM EDTA, pH8.0) and reduced/alkylated using 25mM TCEP solution (Thermo Fisher #77720) and 50mM 2-chloroacetamide (MP Biomedicals #ICN15495580) at 70°C for 20 min. Proteins were recovered by methanol-chloroform precipitation and resuspended in 25mM HEPES (pH8.2) with MS-grade Pierce trypsin (Thermo Fisher #90057) at a 1:50 trypsin:protein weight ratio for 16hrs at 37°C. Peptides were adjusted to 1% trifluoroacetic acid (TFA) and desalted using the StageTip method (Rappsilber, Mann, and Ishihama 2007) and SDBP-RPS material (3M Analytical Biotechnologies). Peptides were washed with 0.2% TFA, and eluted (5% ammonium hydroxide, 80% acetonitrile), followed by drying completely in a SpeedVac (Thermo Fisher) and resuspension in 0.1% formic acid (FA) and 2% acetonitrile (ACN).
Samples were analyzed by nLC-MS in a Dionex Ultimate nRSLC coupled to a Thermo Fisher Q-Exactive HF mass spectrometer using PRM mode. Peptides were separated in a 60 minutes reverse-phase linear gradient (2 to 27% solvent B; solvent A: 0.1% FA, solvent B: 0.1% FA/ 97% ACN) in a PepMap RSLC C19 2μm particle size, 75μm x 50cm EASYSpray column (ThermoFisher ES803) set to 50°C. The instrument was set to 30,000 resolution, 100ms maximum injection time, 5E5 acquisition target, 0.7 m/z isolation window, NCE to 28, and 150m/z fixed first mass.
Created on 3/22/18, 11:18 AM