Summary of Study ST003502
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 PR002148. The data can be accessed directly via it's Project DOI: 10.21228/M8ZR7T 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 | ST003502 |
| Study Title | Quantifying acyl-chain diversity in isobaric compound lipids containing monomethyl branched-chain fatty acids |
| Study Summary | Compound lipids comprise a diverse group of metabolites present in living systems, and metabolic- and environmentally-driven structural distinctions across this family is increasingly linked to biological function. However, methods for deconvoluting these often isobaric lipid species are lacking or require specialized instrumentation. Notably, acyl-chain diversity within cells may be influenced by nutritional states, metabolic dysregulation, or genetic alterations. Therefore, a reliable, validated method of quantifying structurally similar even-, odd-, and branched-chain acyl groups within intact compound lipids will be invaluable for gaining molecular insights into their biological functions. Here we demonstrate the chromatographic resolution of isobaric lipids containing distinct combinations of straight-chain and branched-chain acyl groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry (MS) using a C30 liquid chromatography column. Using metabolically-engineered adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this approach through a combination of fatty acid supplementation and metabolic tracing using monomethyl branched-chain fatty acids and valine. We observe resolution of numerous isobaric triacylglycerols and other compound lipids, demonstrating the resolving utility of this method. This approach strengthens the ability to quantify and characterize the inherent diversity of acyl chains across the lipidome. |
| Institute | Salk Institute for Biological Studies |
| Department | Molecular and Cell Biology |
| Laboratory | Metallo Lab |
| Last Name | Kolar |
| First Name | Matthew |
| Address | 10010 N Torrey Pines Rd |
| mkolar@salk.edu | |
| Phone | 2524534100 |
| Submit Date | 2024-09-20 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-10-18 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002148 |
| Project DOI: | doi: 10.21228/M8ZR7T |
| Project Title: | Quantifying acyl-chain diversity in isobaric compound lipids containing monomethyl branched-chain fatty acids |
| Project Summary: | To assess whether branched-chain fatty acids (BCFAs) could be effectively separated and quantified using a C30 liquid chromatography column, we treated 3T3-L1 cells with a variety of lipid substrates, including odd-chain, even-chain, and branched-chain fatty acids. This initial set of experiments aimed to determine whether retention times for compound lipids shifted with the incorporation of different substrates. Despite identical lipid masses, a shift in retention time would indicate that compound lipids with monomethyl branched fatty acids could be separated. Furthermore, we engineered 3T3-L1 cells lacking branched-keto acid dehydrogenase A (Bckdha) and validated our findings using a deuterated BCFA (iso-FA 16:0[D7]). We observed incorporation of this deuterated BCFA into compound lipids, accompanied by a corresponding shift in retention time. Lipid species were analyzed via UHPLC-MS, confirming that the C30 column successfully separated monomethyl BCFA-containing lipids from their straight-chain counterparts. |
| Institute: | Salk Institute |
| Last Name: | Kolar |
| First Name: | Matthew |
| Address: | 10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA |
| Email: | mkolar@salk.edu |
| Phone: | 8584534100 |
Subject:
| Subject ID: | SU003631 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Cell Strain Details: | 3T3-L1 |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Genotype | Treatment |
|---|---|---|---|---|
| SA385770 | 3T3L1_2HBCFA_BckdhaKO_2 | 3T3-L1 cells | BckdhaKO | iso-FA 16 0[D7] |
| SA385771 | 3T3L1_2HBCFA_BckdhaKO_1 | 3T3-L1 cells | BckdhaKO | iso-FA 16 0[D7] |
| SA385772 | 3T3L1_2HBCFA_BckdhaKO_3 | 3T3-L1 cells | BckdhaKO | iso-FA 16 0[D7] |
| SA385773 | 3T3L1_OCFA_1 | 3T3-L1 cells | WT | FA 15 0 FA 17 0 |
| SA385774 | 3T3L1_OCFA_2 | 3T3-L1 cells | WT | FA 15 0 FA 17 0 |
| SA385775 | 3T3L1_OCFA_3 | 3T3-L1 cells | WT | FA 15 0 FA 17 0 |
| SA385776 | 3T3L1_ECFA_1 | 3T3-L1 cells | WT | FA 16 0 FA16 1(9Z) |
| SA385777 | 3T3L1_ECFA_2 | 3T3-L1 cells | WT | FA 16 0 FA16 1(9Z) |
| SA385778 | 3T3L1_ECFA_3 | 3T3-L1 cells | WT | FA 16 0 FA16 1(9Z) |
| SA385779 | 3T3L1_BCFA_1 | 3T3-L1 cells | WT | iso-FA 16 0 Iso-FA 17 0 anteiso-FA 17 0 |
| SA385780 | 3T3L1_BCFA_2 | 3T3-L1 cells | WT | iso-FA 16 0 Iso-FA 17 0 anteiso-FA 17 0 |
| SA385781 | 3T3L1_BCFA_3 | 3T3-L1 cells | WT | iso-FA 16 0 Iso-FA 17 0 anteiso-FA 17 0 |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO003624 |
| Collection Summary: | Cells were washed with cold PBS and afterwards subjected to lipid extraction. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003640 |
| Treatment Summary: | WT 3T3-L1 cells were treated with 100 uM of the respective fatty acid mixture of ECFAs vs BCFAs or OCFAs for 96 hours prior to lipid extraction. The bckdhaKO 3T3-L1 cells were treated with 100 uM of iso-FA 16:0[D7]-BSA complex for 96 hours prior to lipid extraction. These cells with the iso-FA 16:0[D7] were also treated with cobalamin 500 nM. |
Sample Preparation:
| Sampleprep ID: | SP003638 |
| Sampleprep Summary: | Lipid extraction was carried out using a modified Folch methanol/chloroform/water extraction at a ratio of 5:5:2 with the inclusion of 10 nM FA 12:0 dodecylglycerol, 10 nM palmitate[D31], 10 ng of each lipid in the Avanti EquiSPLASH mix. The methanol phase was washed a second time with chloroform after addition of 2μL formic acid. The chloroform phase (bottom layer) was transferred and dried under nitrogen gas at room temperature. Samples were stored at -20°C before analysis by LC-MS/MS. |
Combined analysis:
| Analysis ID | AN005750 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Accucore C30 Column (250 x 2.1 mm, 2.6 µm) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE |
| Units | AUC |
Chromatography:
| Chromatography ID: | CH004364 |
| Chromatography Summary: | The LC gradient ran from 30% to 43% B from 3-8 min, then from 43% to 50% B from 8-9 min, then 50 to 90% B from 9-18 min, then 90 to 99% B from 18-26 min, then held at 99% B from 26-30 min, before returning to 30% B in 6 min and held for a further 4 min |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Accucore C30 Column (250 x 2.1 mm, 2.6 µm) |
| Column Temperature: | 40°C |
| Flow Gradient: | 0 min, 30%B; 3-8 min, 30-43%B; 8-9 min, 43-50%B; 9-18 min, 50-90%B, 18-26 min 90-99%B, 26-30 min, 99%B, 30-40 min, 30% B |
| Flow Rate: | 0.2 mL/min |
| Solvent A: | 40% Water/60% Acetonitrile; 10 mM Ammonium formate, 0.1% Formic acid |
| Solvent B: | 10% Acetonitrile/90% 2-propanol; 10 mM ammonium formate, 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005473 |
| Analysis ID: | AN005750 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | El-MAVEN TGs, phospholipids, and sphingolipids were analyzed in positive mode using spray voltage 3.2 kV. Sweep gas flow was 1 arbitrary units, auxiliary gas flow 2 arbitrary units and sheath gas flow 40 arbitrary units, with a capillary temperature of 325°C. Full MS (scan range 200-2000 m/z) was used at 70,000 resolution with 1e6 automatic gain control and a maximum injection time of 100 ms. Data dependent MS2 (Top 6) mode at 17,500 resolution with automatic gain control set at 1e5 with a maximum injection time of 50 ms was used. Extracted ion chromatograms for each analyzed lipid was generated using a m/z + 5 ppm mass window around the calculated exact mass. |
| Ion Mode: | POSITIVE |
