Summary of Study ST004003
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 PR002508. The data can be accessed directly via it's Project DOI: 10.21228/M8FZ6V This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST004003 |
| Study Title | Measuring de novo PC synthesis in HCMV infected fibroblasts using 13C-choline. |
| Study Summary | The host de novo PC pathway is the canonical pathway for PC synthesis. We determined if the de novo PC pathway was active in HCMV infection. Our findings demonstrate that infection promotes the de novo PC pathway to increase PC lipids. |
| 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 | 2025-06-22 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-06-27 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002508 |
| Project DOI: | doi: 10.21228/M8FZ6V |
| Project Title: | Measuring de novo PC synthesis in HCMV infected fibroblasts using 13C-choline. |
| 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: | SU004141 |
| 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 | Time (hpi) | Infection status |
|---|---|---|---|---|
| SA461434 | 20200204_pos_13C_choline_TB40E_24_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 24 | HCMV |
| SA461435 | 20200204_pos_13C_choline_TB40E_24_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 24 | HCMV |
| SA461436 | 20200225_MS1_280k_pos_C13_choline_CMV_24B | Primary human fibroblast cells (HFF) | 24 | HCMV |
| SA461437 | 20200225_MS1_280k_pos_C13_choline_CMV_24A | Primary human fibroblast cells (HFF) | 24 | HCMV |
| SA461438 | 20200206_pos_13C_choline_TB40E_24_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 24 | HCMV |
| SA461439 | 20200206_pos_13C_choline_TB40E_24_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 24 | HCMV |
| SA461440 | 20200206_pos_13C_choline_mock_24_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 24 | Uninfected |
| SA461441 | 20200225_MS1_280k_pos_C13_choline_Mock_24B | Primary human fibroblast cells (HFF) | 24 | Uninfected |
| SA461442 | 20200225_MS1_280k_pos_C13_choline_Mock_24A | Primary human fibroblast cells (HFF) | 24 | Uninfected |
| SA461443 | 20200204_pos_13C_choline_mock_24_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 24 | Uninfected |
| SA461444 | 20200204_pos_13C_choline_mock_24_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 24 | Uninfected |
| SA461445 | 20200206_pos_13C_choline_mock_24_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 24 | Uninfected |
| SA461446 | 20200206_pos_13C_choline_TB40E_48_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 48 | HCMV |
| SA461447 | 20200225_MS1_280k_pos_C13_choline_CMV_48B | Primary human fibroblast cells (HFF) | 48 | HCMV |
| SA461448 | 20200225_MS1_280k_pos_C13_choline_CMV_48A | Primary human fibroblast cells (HFF) | 48 | HCMV |
| SA461449 | 20200204_pos_13C_choline_TB40E_48_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 48 | HCMV |
| SA461450 | 20200204_pos_13C_choline_TB40E_48_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 48 | HCMV |
| SA461451 | 20200206_pos_13C_choline_TB40E_48_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 48 | HCMV |
| SA461452 | 20200206_pos_13C_choline_mock_48_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 48 | Uninfected |
| SA461453 | 20200225_MS1_280k_pos_C13_choline_Mock_48B | Primary human fibroblast cells (HFF) | 48 | Uninfected |
| SA461454 | 20200225_MS1_280k_pos_C13_choline_Mock_48A | Primary human fibroblast cells (HFF) | 48 | Uninfected |
| SA461455 | 20200206_pos_13C_choline_mock_48_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 48 | Uninfected |
| SA461456 | 20200204_pos_13C_choline_mock_48_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 48 | Uninfected |
| SA461457 | 20200204_pos_13C_choline_mock_48_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 48 | Uninfected |
| SA461458 | 20200204_pos_13C_choline_TB40E_72_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 72 | HCMV |
| SA461459 | 20200206_pos_13C_choline_TB40E_72_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 72 | HCMV |
| SA461460 | 20200206_pos_13C_choline_TB40E_72_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 72 | HCMV |
| SA461461 | 20200204_pos_13C_choline_TB40E_72_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 72 | HCMV |
| SA461462 | 20200225_MS1_280k_pos_C13_choline_CMV_72A | Primary human fibroblast cells (HFF) | 72 | HCMV |
| SA461463 | 20200225_MS1_280k_pos_C13_choline_CMV_72B | Primary human fibroblast cells (HFF) | 72 | HCMV |
| SA461464 | 20200206_pos_13C_choline_mock_72_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 72 | Uninfected |
| SA461465 | 20200206_pos_13C_choline_mock_72_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 72 | Uninfected |
| SA461466 | 20200204_pos_13C_choline_mock_72_b_MS1only140Kres | Primary human fibroblast cells (HFF) | 72 | Uninfected |
| SA461467 | 20200225_MS1_280k_pos_C13_choline_Mock_72a | Primary human fibroblast cells (HFF) | 72 | Uninfected |
| SA461468 | 20200225_MS1_280k_pos_C13_choline_Mock_72B | Primary human fibroblast cells (HFF) | 72 | Uninfected |
| SA461469 | 20200204_pos_13C_choline_mock_72_a_MS1only140Kres | Primary human fibroblast cells (HFF) | 72 | Uninfected |
| Showing results 1 to 36 of 36 |
Collection:
| Collection ID: | CO004134 |
| 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 soluition |
| 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: | TR004150 |
| Treatment Summary: | Cells were grown to full confluence and held for 3-days in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS). 24 hours prior to infection, cells were starved of serum in DMEM lacking FBS. Cells were HCMV-infected or mock-infected in serum-free DMEM for 1 hour. 1 hpi, 13C-choline isotopic tracer was fed to cells in serum-free growth medium we previously prepared (based on the published DMEM Gibco recipe). Tracer media contains 13C-choline in place 12C-choline. Tracer molecule was added at the same concentration non-tracer molecule is normally present in DMEM (13C-choline (4mg/L)). Medium was replaced at 48 hpi. |
Sample Preparation:
| Sampleprep ID: | SP004147 |
| 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 -80℃ 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℃. |
Chromatography:
| Chromatography ID: | CH005012 |
| 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 |
Analysis:
| Analysis ID: | AN006602 |
| Analysis Type: | MS |
| Analysis Protocol File: | CLM-548-0.1_CHOLINE+CHLORIDE+(1,2-13C2,+99%)+-+CLM-548+-+V.3.1+-+US+-+English+US.pdf |
| Chromatography ID: | CH005012 |
| Num Factors: | 6 |
| Num Metabolites: | 276 |
| Units: | Peak Area |
