Summary of Study ST003377
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 PR002085. The data can be accessed directly via it's Project DOI: 10.21228/M83R6P 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 | ST003377 |
| Study Title | Sphingolipid secretory flux from Huh7 SPTLC3 KO cells |
| Study Summary | We analyzed 13C labeling on sphingolipids in fresh and spent media from Huh7 control and SPTLC3 (serine palmitoyltransferase) KO (Knockout) cells treated with BSA-palmitate and the stable isotope tracers [13C]serine and [13C]glycine to measure sphingolipid secretory flux. We aimed to determine if a knockout of SPTLC3 could reduce the secretion of SM from Huh7 cells. This mechanism could provide insight on how hepatic SPT flux is linked to VLDL secretion and the circulation of atherogenic lipoproteins that contain sphingolipids like SM. |
| Institute | Salk Institute for Biological Studies |
| Last Name | Gengatharan |
| First Name | Jivani |
| Address | 10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA |
| jivani14@gmail.com | |
| Phone | (858) 453-4100 |
| Submit Date | 2024-08-02 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-08-12 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002085 |
| Project DOI: | doi: 10.21228/M83R6P |
| Project Title: | Altered sphingolipid biosynthetic flux and lipoprotein trafficking contribute to trans fat-induced atherosclerosis |
| Project Summary: | The goal of the project is to determine the role of sphingolipid metabolism in atherosclerosis induced by dietary trans fat. We analyzed lipid metabolites in Huh7 cells following various fatty acid treatments, with specific focus on cis and trans unsaturated fatty acids. Additionally, we analyzed lipid metabolites in plasma and liver of Ldlr-/- mice fed high-fat diets enriched in cis or trans fatty acids in the presence or absence of myriocin, a pharmacological inhibitor of Serine palmitoyltransferase (SPT), the initial rate-limiting enzyme of sphingolipid biosynthesis. |
| Institute: | Salk Institute for Biological Studies |
| Last Name: | Gengatharan |
| First Name: | Jivani |
| Address: | 10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA |
| Email: | jivani14@gmail.com |
| Phone: | (858) 453-4100 |
Subject:
| Subject ID: | SU003498 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Strain Details: | Huh7 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Condition |
|---|---|---|---|
| SA367148 | Huh7spentmedia_delip_AAVS_1 | Huh7 media | AAVS1 Control Spent Media |
| SA367149 | Huh7spentmedia_delip_AAVS_2 | Huh7 media | AAVS1 Control Spent Media |
| SA367150 | Huh7spentmedia_delip_AAVS_3 | Huh7 media | AAVS1 Control Spent Media |
| SA367151 | Huh7spentmedia_delip_AAVS_2_MS2 | Huh7 media | AAVS1 Control Spent Media |
| SA367152 | Huh7freshmedia_delip_1 | Huh7 media | Fresh Media |
| SA367153 | Huh7freshmedia_delip_2 | Huh7 media | Fresh Media |
| SA367154 | Huh7freshmedia_delip_3 | Huh7 media | Fresh Media |
| SA367155 | Huh7freshmedia_delip_3_MS2 | Huh7 media | Fresh Media |
| SA367156 | Huh7spentmedia_delip_SPT3KO1_1 | Huh7 media | SPTLC3 KO #1 Spent Media |
| SA367157 | Huh7spentmedia_delip_SPT3KO1_2 | Huh7 media | SPTLC3 KO #1 Spent Media |
| SA367158 | Huh7spentmedia_delip_SPT3KO1_3 | Huh7 media | SPTLC3 KO #1 Spent Media |
| SA367159 | Huh7spentmedia_delip_SPT3KO2_1 | Huh7 media | SPTLC3 KO #2 Spent Media |
| SA367160 | Huh7spentmedia_delip_SPT3KO2_2 | Huh7 media | SPTLC3 KO #2 Spent Media |
| SA367161 | Huh7spentmedia_delip_SPT3KO2_3 | Huh7 media | SPTLC3 KO #2 Spent Media |
| Showing results 1 to 14 of 14 |
Collection:
| Collection ID: | CO003491 |
| Collection Summary: | To calculate secretory flux, 0.75 mL of fresh media or 1.5 mL of spent media was evaporated under vacuum at 4°C and resuspended into 0.1 mL of H2O. |
| Sample Type: | Culture Media |
Treatment:
| Treatment ID: | TR003507 |
| Treatment Summary: | To measure sphingomyelin secretory flux, cells were treated with 100 µM BSA-palmitate and traced with [13C3]serine and [13C2]glycine for 48 hours in delipidated media to increase the percentage of labeled lipids in spent media. |
Sample Preparation:
| Sampleprep ID: | SP003505 |
| Sampleprep Summary: | Media was spiked with the following internal standards: 20 pmol sphinganine-d7 (Avanti Polar Lipids, Cat# 860658), deoxysphinganine-d3 (Avanti Polar Lipids, Cat# 860474), 100 pmol d18:0-d7/13:0 dihydroceramide (Avanti Polar Lipids, Cat# 330726), 200 pmol d18:1-d7/15:0 ceramide (Avanti Polar Lipids, Cat# 860681), 100 pmol d18:1-d7/15:0 glucosylceramide (Avanti Polar Lipids, Cat# 330729), 100 pmol d18:1-d7/15:0 lactosylceramide (Avanti Polar Lipids, Cat# 330727), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), and 200 pmol sphingomyelin (d18:1/18:1)-d9 (Avanti Polar Lipids, Cat#860740). 0.5 mL methanol, 0.5 mL H2O, and 1 mL chloroform were added directly. Samples were vortexed for 5 min and centrifuged for 5 min at 4°C at 15,000g. The organic phase was collected and 2 μL of formic acid was added to the remaining polar phase which was re-extracted with 1 mL of chloroform. Combined organic phases were dried under nitrogen. After dried extracts for media were resuspended in 60 µl MeOH, 5 µL of sample was injected. |
Combined analysis:
| Analysis ID | AN005529 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH004205 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 35°C |
| Flow Gradient: | 0 min, 30%B; 1 min, 30%B; 2 min, 70%B; 11 min, 95%B; 17 min, 30%B; 21.5 min, 30%B; 27 min, 30%B |
| Flow Rate: | 0.2 mL/min |
| Solvent A: | 98% water/2% methanol; 5 mM ammonium acetate |
| Solvent B: | 50% methanol/50% isopropanol; 5 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005254 |
| Analysis ID: | AN005529 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Lipids were identified and quantified using EL-MAVEN using exact mass of precursor ion in MS1 chromatogram and product ion in MS2 spectra. The percent isotopologue distribution of each sphingolipid was determined and corrected for natural 13C abundance using in-house algorithms adapted from a previous report. |
| Ion Mode: | POSITIVE |
