Summary of Study ST003382
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 | ST003382 |
| Study Title | Sphingolipid biosynthetic flux in Huh7 SPTLC3 KO cells |
| Study Summary | We analyzed 13C labeling on sphingolipids in 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 intracellular sphingolipid biosynthetic flux. 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 SPT (serine palmitoyltransferase), 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 |
| 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: | SU003503 |
| 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 |
|---|---|---|---|
| SA367342 | Huh7cells_AAVS1_1 | Huh7 cells | AAVS1 Control |
| SA367343 | Huh7cells_AAVS1_2 | Huh7 cells | AAVS1 Control |
| SA367344 | Huh7cells_AAVS1_3 | Huh7 cells | AAVS1 Control |
| SA367345 | Huh7cells_AAVS1_1_MS2 | Huh7 cells | AAVS1 Control |
| SA367346 | Huh7cells_AAVS1_2_MS2 | Huh7 cells | AAVS1 Control |
| SA367347 | Huh7cells_SPT3KO1_1 | Huh7 cells | SPTLC3 KO #1 |
| SA367348 | Huh7cells_SPT3KO1_2 | Huh7 cells | SPTLC3 KO #1 |
| SA367349 | Huh7cells_SPT3KO1_3 | Huh7 cells | SPTLC3 KO #1 |
| SA367350 | Huh7cells_SPT3KO2_1 | Huh7 cells | SPTLC3 KO #2 |
| SA367351 | Huh7cells_SPT3KO2_2 | Huh7 cells | SPTLC3 KO #2 |
| SA367352 | Huh7cells_SPT3KO2_3 | Huh7 cells | SPTLC3 KO #2 |
| Showing results 1 to 11 of 11 |
Collection:
| Collection ID: | CO003496 |
| Collection Summary: | Cells (~400,000 cells) were 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). Cells were scraped with 0.5 mL methanol and 0.5 mL H2O. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003512 |
| Treatment Summary: | To measure intracellular sphingolipid biosynthetic flux in Huh7 control and SPTLC3 KO cell lines, we treated cells with 100 µM BSA-palmitate and performed stable isotope tracing with a combination [13C3]serine and [13C2]glycine for 48 hours. |
Sample Preparation:
| Sampleprep ID: | SP003510 |
| Sampleprep Summary: | Cells (~400,000 cells) were 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). Cells were scraped with 0.5 mL methanol and 0.5 mL H2O. Homogenate aliquot of 100 µL was taken to determine protein content using the BCA protein assay (Thermo Fisher Scientific). The remaining homogenate was transferred to a new Eppendorf tube and 1 mL chloroform was added. For plasma or media, 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 cells were resuspended in 60 µL MeOH, 5 µL of sample was injected. |
Combined analysis:
| Analysis ID | AN005540 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 (100 x 2.1mm,1.7µm) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH004212 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (100 x 2.1mm,1.7µm) |
| 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: | MS005265 |
| Analysis ID: | AN005540 |
| 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 |
