Summary of Study ST004000
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002505. The data can be accessed directly via it's Project DOI: 10.21228/M8V54W This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST004000 |
| Study Title | Measuring PC lipids in HCMV infected fibroblasts treated with phosphonoacetic acid (PAA). |
| Study Summary | HCMV viral gene expression is classified into three kinetic classes, immediate early (IE), early (E) and late genes (L). HCMV promotes PC lipids by 48 hpi relative to uninfected cells, suggesting the involvement of an IE or E viral protein in regulating PC lipid levels. Viral DNA synthesis initiates L viral gene expression. Phosphonoacetic acid (PAA) inhibits HCMV genome replication and consequentially, L gene expression. Therefore, we used PAA to test whether L gene expression was required to reprogram PC lipid levels in HCMV infection. We find that PAA-treated cells contain similar levels of PC lipids to vehicle-treated conditions thus, L HCMV gene expression is not required to promote PC levels. |
| Institute | University of Arizona |
| Department | Immunobiology |
| Laboratory | John G. Purdy, PhD |
| Last Name | Kline |
| First Name | Ian |
| Address | 1657 E Helen St, Tucson, AZ 85721 |
| ikline@arizona.edu | |
| Phone | 5209092596 |
| Submit Date | 2025-06-17 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-06-25 |
| Release Version | 1 |
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Project:
| Project ID: | PR002505 |
| Project DOI: | doi: 10.21228/M8V54W |
| Project Title: | Measuring PC lipids in HCMV infected fibroblasts treated with phosphonoacetic acid (PAA). |
| Project Summary: | Human cytomegalovirus (HCMV) is a common herpesvirus that establishes a lifelong and persistent infection in its human host. HCMV infection in most people does not cause overt disease. However, in immunocompromised individuals, severe CMV-associated disease can lead to permanent disabilities and even death. Additionally, congenital CMV is the leading infectious cause of birth defects. Viruses have evolved to hijack host metabolic pathways to facilitate their replication cycle. We previously reported HCMV infection increases phosphatidylcholine (PC) lipid levels, including PCs with VLCFAs. To expand upon the previously reported PC phenotype in HCMV infection, we determined the PC lipidome of several infected cell types grown under various growth conditions. Additionally, we determined which host pathways HCMV reprograms to induce PC lipid synthesis and describe when during infection PC lipids changes occur. |
| Institute: | University of Arizona |
| Department: | Immunobiology |
| Laboratory: | John G. Purdy, PhD |
| Last Name: | Kline |
| First Name: | Ian |
| Address: | 1657 E Helen St, Tucson, AZ 85721 |
| Email: | ikline@arizona.edu |
| Phone: | 5209092596 |
| Funding Source: | National Institute of Health (NIH) National Institute of Allergy and Infectious Disease (NIAID) R01AI162671, R01AI155539, F32AI178919, and National Institute of Aging (NIA) T32AG058503 award. |
Subject:
| Subject ID: | SU004138 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male |
| Cell Strain Details: | HFF |
| Cell Passage Number: | <30 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | DNA synthesis inhibtor |
|---|---|---|---|
| SA461354 | 20241023_pos_HCMV_h2O_72_R1_a | Primary human fibroblast cells (HFF) | H20 |
| SA461355 | 20241023_pos_HCMV_h2O_72_R1_b | Primary human fibroblast cells (HFF) | H20 |
| SA461356 | 20241023_pos_HCMV_h2O_72_R2_a | Primary human fibroblast cells (HFF) | H20 |
| SA461357 | 20241023_pos_HCMV_h2O_72_R2_b | Primary human fibroblast cells (HFF) | H20 |
| SA461358 | 20241023_pos_HCMV_h2O_72_R3_a | Primary human fibroblast cells (HFF) | H20 |
| SA461359 | 20241023_pos_HCMV_h2O_72_R3_b | Primary human fibroblast cells (HFF) | H20 |
| SA461360 | 20241023_pos_HCMV_PAA_72_R1_a | Primary human fibroblast cells (HFF) | PAA 100ug/mL |
| SA461361 | 20241023_pos_HCMV_PAA_72_R1_b | Primary human fibroblast cells (HFF) | PAA 100ug/mL |
| SA461362 | 20241023_pos_HCMV_PAA_72_R2_a | Primary human fibroblast cells (HFF) | PAA 100ug/mL |
| SA461363 | 20241023_pos_HCMV_PAA_72_R2_b | Primary human fibroblast cells (HFF) | PAA 100ug/mL |
| SA461364 | 20241023_pos_HCMV_PAA_72_R3_a | Primary human fibroblast cells (HFF) | PAA 100ug/mL |
| SA461365 | 20241023_pos_HCMV_PAA_72_R3_b | Primary human fibroblast cells (HFF) | PAA 100ug/mL |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO004131 |
| Collection Summary: | General Notes: -Work over ice when possible (during scraping and between vortex steps etc.). -Chloroform leeches plastics. Avoid contact with plastic caps, tubes and gloves around glass vial tops. -Work in manageable batch numbers. A batch of 8-16 samples at a time is common. -Clean syringes using chloroform before and after collection. Use a unique syringe for each sample (the same syringe can be used if A/B technical replicates are used. Recommend a quick flush of the syringe using 500 μL chloroform before moving from A to B). Begin by counting cell numbers for each sample using a dedicated well meant for MS normalization based on cell count. In a 6-well plate, wash cells 2x with cold PBS. Add 1mL of cold 50% methanol to each well and scrape cells into glass vials. Add 500 μL of chloroform. Vortex on low setting (careful to avoid splashing chloroform onto plastic caps and liners). Centrifuge @1000g for 5 mins. There should be a clear phase separation of methanol and cell debris on top, while the lower phase contains chloroform and lipids. Use a syringe to carefully extract the lower phase without transferring cell debris from the top layer. Transfer to clean vial and place on ice. Once all lipids have been extracted, add 500 μL of chloroform and repeat the process again once more. In total each sample should contain ~1 mL of chloroform and lipids from two extractions. Carefully, dry lipids under nitrogen gas. Avoid direct high pressure air flow onto chloroform:lipid solution as it can splash high up in vial walls. Store at -80℃ for up to 30 days. Samples may become unstable and degrade over time. |
| Sample Type: | Cultured cells |
| Collection Location: | Fume hood required when working with chloroform. |
| Collection Frequency: | Perform extraction twice on each sample. |
| Collection Duration: | 1-2 hours dependent upon batch size |
| Volumeoramount Collected: | ~1 mL chloroform:lipid solution |
| Storage Conditions: | -80℃ |
| Collection Vials: | Glass vials, no plastic! (plastic cap is okay) |
| Storage Vials: | Glass vials, no plastic! (plastic cap is okay) |
| Collection Tube Temp: | 25 |
| Additives: | UHPLC 100% chloroform |
Treatment:
| Treatment ID: | TR004147 |
| Treatment Summary: | Cells were grown to full confluence in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS). Cells were growth arrested at full confluence for 3 days and then switched to serum-free DMEM for 24 hours. Cells were HCMV-infected for 1 hour in serum-free DMEM. At 1 hpi, cells were washed of unattached virus and maintained in serum-free DMEM with either 100ug/mL PAA or vehicle H2O. PAA or H2O was replaced every 24 h and medium was refreshed at 48 hpi. |
Sample Preparation:
| Sampleprep ID: | SP004144 |
| Sampleprep Summary: | Before starting, calculate the volume of 1:1:1 choloroform:methanol:isopropanol resuspension buffer needed for each sample. Volume of 1:1:1 is dependent on normalize cell count from experiment. Use 200 μL of 1:1:1 for every 2E5 cells. When ready to start resuspension, remove dried lipids from -80C storage and resuspend in volume of 1:1:1 solution calculated for each sample. No cell conditions use 200 μL. Use gentle vortex to allow for dried lipids from vial wall to get into solution. Prepare "blank" vials of 1:1:1 for buffer background analysis. Store lipids in autosampler between 4-7℃. |
Combined analysis:
| Analysis ID | AN006595 |
|---|---|
| Chromatography ID | CH005007 |
| MS ID | MS006294 |
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 (100 x 2.1 mm, 2.6 μm) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Orbitrap Exploris 240 |
| Ion Mode | POSITIVE |
| Units | Peak Area |
Chromatography:
| Chromatography ID: | CH005007 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (100 x 2.1 mm, 2.6 μm) |
| Column Temperature: | 60℃ |
| Flow Gradient: | 75% solvent A–25% solvent B for 2 min, 35% solvent A–65% solvent B for 2min at a curve value of 4, a hold at 35% solvent A–65% solvent B for 1min, 0% solvent A–100% solvent B for 11min at a curve value of 4, and a hold at 0% solvent A–100% solvent B for 4 min. |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 40% Water/60% Methanol; 10mM Ammonium formate; 0.1% Formic acid |
| Solvent B: | 10% Methanol/90% Isopropanol; 10mM Ammonium formate; 0.1% Formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS006294 |
| Analysis ID: | AN006595 |
| Instrument Name: | Thermo Orbitrap Exploris 240 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | PCs were identified in positive mode using parent lipid m/z, RT and choline head fragment of 184.073 m/z. Confirmation of PCs was performed by FA tail analysis in negative mode. |
| Ion Mode: | POSITIVE |
