Summary of Study ST001359
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 PR000929. The data can be accessed directly via it's Project DOI: 10.21228/M8M98M 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 | ST001359 |
| Study Title | Monophasic lipidomics extraction in cancer cell line |
| Study Type | Lipidomics |
| Study Summary | We performed a comprehensive characterization of a monophasic extraction method in cancer cell lines. We used pharmacological perturbation on HepG2 cells to identify changes in different lipid families. We optimized the MS parameters, the chromatography and the data analysis to perform rapid and robust lipidomics analysis from cell lines. |
| Institute | Beatson Institute for Cancer Research |
| Department | Metabolomics |
| Laboratory | Metabolomics |
| Last Name | Rodriguez Blanco |
| First Name | Giovanny |
| Address | Garscube State, Switchback road, Glasgow |
| g.blanco@ed.ac.uk | |
| Phone | +447526056849 |
| Submit Date | 2020-03-26 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2020-09-29 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR000929 |
| Project DOI: | doi: 10.21228/M8M98M |
| Project Title: | Monophasic lipidomics extraction in cancer cell lines |
| Project Type: | Lipidomics |
| Project Summary: | We performed a comprehensive characterization of a monophasic extraction method in cancer cell lines. We used pharmacological perturbation on HepG2 cells to identify changes in different lipid families. We optimized the MS parameters, the chromatography and the data analysis to perform rapid and robust lipidomics analysis from cell lines. |
| Institute: | Institute of Genetics and Molecular Medicine |
| Laboratory: | Mass Spec Lab |
| Last Name: | Rodriguez Blanco |
| First Name: | Giovanny |
| Address: | Crewe Road South, Edinburgh, Midlothian, EH42XU, United Kingdom |
| Email: | g.blanco@ed.ac.uk |
| Phone: | 00447526056849 |
Subject:
| Subject ID: | SU001433 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | HPACC |
| Cell Passage Number: | 10-20 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Variable |
|---|---|---|
| SA098893 | VV_13_HEpG2_C1 | Control |
| SA098894 | VV_15_HEpG2_C3 | Control |
| SA098895 | VV_14_HEpG2_C2 | Control |
| SA098896 | VV_18_HEpG2_SDC3 | SDC_i |
| SA098897 | VV_16_HEpG2_SDC1 | SDC_i |
| SA098898 | VV_17_HEpG2_SDC2 | SDC_i |
| Showing results 1 to 6 of 6 |
Collection:
| Collection ID: | CO001428 |
| Collection Summary: | Single-phase lipid extraction was performed according to some reported protocols for metabolomics extraction (MacKay et al., 2015). Briefly, media was quickly removed from the plates and each well was washed twice with ice-cold PBS. An extracting solution containing either isopropanol (IPA) or a mixture 1:1 of methanol-butanol (BuMe), kept at 4°C, was used to simultaneously quench all metabolic reactions and to extract intra-cellular lipids. Plates were incubated on dry ice for 20 min and extracted lipids were transferred to 1.5 ml Eppendorf tubes (Stevenage, UK) followed by centrifugation (4°C) at 14000 rpm for 10min to remove the denatured proteins. Supernatants were collected and stored at -80°C prior to LC-MS analysis. Protein pellets attached to the plate were left to dry overnight for the subsequent protein normalisation by the modified-Lowry procedure (MacKay et al., 2015). |
| Collection Protocol Filename: | garodriguezblanco10_20200326_110242_PR_CO_Methods.docx |
| Sample Type: | HepG2 cells |
Treatment:
| Treatment ID: | TR001448 |
| Treatment Summary: | HepG2 cells were treated with a desaturase inhibitor for 24h. |
Sample Preparation:
| Sampleprep ID: | SP001441 |
| Sampleprep Summary: | Single-phase lipid extraction was performed according to some reported protocols for metabolomics extraction (MacKay et al., 2015). Briefly, media was quickly removed from the plates and each well was washed twice with ice-cold PBS. An extracting solution containing either isopropanol (IPA) or a mixture 1:1 of methanol-butanol (BuMe), kept at 4°C, was used to simultaneously quench all metabolic reactions and to extract intra-cellular lipids. Plates were incubated on dry ice for 20 min and extracted lipids were transferred to 1.5 ml Eppendorf tubes (Stevenage, UK) followed by centrifugation (4°C) at 14000 rpm for 10min to remove the denatured proteins. Supernatants were collected and stored at -80°C prior to LC-MS analysis. Protein pellets attached to the plate were left to dry overnight for the subsequent protein normalisation. |
Combined analysis:
| Analysis ID | AN002263 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Ultimate 3000 |
| Column | Waters Acquity CSH 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 | Normalised peak area |
Chromatography:
| Chromatography ID: | CH001662 |
| Chromatography Summary: | The mobile phases consisted of 60:40 ACN: H2O with 10mM ammonium formate, 0.1% formic acid and 5µM of phosphoric acid (A) and 90:10 IPA:ACN with 10 mM ammonium formate, 0.1% formic acid and 5µM phosphoric acid (B). The gradient was as follows: 0-2 min 30% (B); 2-8 min 50% (B); 8-15 min 99% (B), 15-16 min 99% (B), 16-17 min 30% (B). Sample temperature was maintained at 6°C in the autosampler and 5 µL of sample were injected into the LC-MS instrument. |
| Instrument Name: | Ultimate 3000 |
| Column Name: | Waters Acquity CSH C18 (100 x 2.1mm,1.7um) |
| Flow Gradient: | 0-2 min 30% (B); 2-8 min 50% (B); 8-15 min 99% (B), 15-16 min 99% (B), 16-17 min 30% (B). |
| Solvent A: | 60% acetonitrile/40% water; 0.1% formic acid; 10mM ammonium formate; 5µM phosphoric acid |
| Solvent B: | 90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate;5µM phosphoric acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002107 |
| Analysis ID: | AN002263 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Thermo Q-Exactive Orbitrap MS instrument was operated in both positive and negative polarities, using the following parameters: mass range 240-1200 m/z (positive) and 240-1600 (negative), spray voltage 3.8kV (ESI+) and 3kV (ESI-), sheath gas (nitrogen) flow rate 60 units, auxiliary gas (nitrogen) flow rate 25 units, capillary temperature (320°C), full scan MS1 mass resolving power 70,000. Data dependent fragmentation (dd-MS/MS) parameters for each polarity as follows: TopN: 10, resolution 17,500 units, maximum injection time: 25 ms, automatic gain control target: 5e5 and normalised collision energy of 20 and 25 (arbitrary units) in positive polarity. TopN: 5, resolution 17,500 units, maximum injection time: 80 ms automatic gain control target: 5e5 and normalised collision energy of 20 and 30 (arbitrary units) in negative polarity. The instrument was externally calibrated to <1ppm using ESI positive and negative calibration solutions (Thermo Fisher Scientific). |
| Ion Mode: | POSITIVE |
