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.

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Study IDST001359
Study TitleMonophasic lipidomics extraction in cancer cell line
Study TypeLipidomics
Study SummaryWe 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
DepartmentMetabolomics
LaboratoryMetabolomics
Last NameRodriguez Blanco
First NameGiovanny
AddressGarscube State, Switchback road, Glasgow
Emailg.blanco@ed.ac.uk
Phone+447526056849
Submit Date2020-03-26
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2020-09-29
Release Version1
Giovanny Rodriguez Blanco Giovanny Rodriguez Blanco
https://dx.doi.org/10.21228/M8M98M
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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
SA098893VV_13_HEpG2_C1Control
SA098894VV_15_HEpG2_C3Control
SA098895VV_14_HEpG2_C2Control
SA098896VV_18_HEpG2_SDC3SDC_i
SA098897VV_16_HEpG2_SDC1SDC_i
SA098898VV_17_HEpG2_SDC2SDC_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)
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
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