Summary of study ST001265

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 PR000850. The data can be accessed directly via it's Project DOI: 10.21228/M8T98G This work is supported by NIH grant, U2C- DK119886.

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Study IDST001265
Study TitleComparative metabolomics of MCF-7 breast cancer cells using different extraction solvents assessed by mass spectroscopy
Study TypeAnalysing metabolomics using GC Mass Spectroscopy
Study SummaryMetabolic profiling of cancer cells can play a vital role in revealing the molecular bases of cancer development and progression. In this study, gas chromatography coupled with mass spectrometry (GC-MS) was employed for the determination of signatures found in ER+/ PR+ breast cancer cells derived from MCF-7 using different extraction solvents including: A, formic acid in water; B, ammonium hydroxide in water; C, ethyl acetate; D, methanol: water (1:1, v/v); and E, acetonitrile: water (1:1, v/v). The greatest extraction rate and diversity of metabolites occurs with extraction solvents A and E. Extraction solvent D showed moderate extraction efficiency, whereas extraction solvent B and C showed inferior metabolite diversity. Metabolite set enrichment analysis results showed energy production pathways to be key in MCF-7 cell lines. This study showed that mass spectrometry could identify key metabolites associated with cancers. The highest enriched pathways were related to energy production as well as Warburg effect pathways, which may shed light on how energy metabolism has been hijacked to encourage tumour progression and eventually metastasis in breast cancer.
Institute
Sharjah Institute for Medical Research
DepartmentClinical Science
Last NameHamoudi
First NameRifat
AddressCollege of Medicine, University of Sharjah
Emailrhamoudi@sharjah.ac.ae
Phone567154756
Submit Date2019-07-08
Total SubjectsFive different extractions
Study CommentsMCF-7 cell line
Raw Data AvailableYes
Raw Data File Type(s).qgd
Analysis Type DetailGC-MS
Release Date2019-10-11
Release Version1
Rifat Hamoudi Rifat Hamoudi
https://dx.doi.org/10.21228/M8T98G
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000850
Project DOI:doi: 10.21228/M8T98G
Project Title:Comparative metabolomics of MCF-7 breast cancer cells using different extraction solvents assessed by mass spectroscopy
Project Type:Metabolomics using Mass Spectrometry
Project Summary:Metabolic profiling of cancer cells can play an important role in revealing the molecular bases of cancer development and progression. In this work, gas chromatography coupled with mass spectrometry (GC-MS) was employed for the determination of signatures found in ER+/PR+ breast cancer cells derived from MCF-7 using different extraction solvents including: A, formic acid in water; B, ammonium hydroxide in water; C, ethyl acetate; D, methanol: water (1:1, v/v); and E, acetonitrile: water (1:1, v/v). The greatest extraction rate and diversity of metabolites occurs with extraction solvents A and E. Extraction solvent D showed moderate extraction efficiency, whereas extraction solvent B and C showed inferior metabolite diversity. Metabolite set enrichment analysis results showed energy production pathways to be key in MCF-7 cell lines. This study showed that mass spectrometry could identify key metabolites associated with cancers. The highest enriched pathways were related to energy production as well as Warburg effect pathways, which may shed light on how energy metabolism has been hijacked to encourage tumour progression and eventually metastasis in breast cancer.
Institute:University of Sharjah
Department:Sharjah Institute for Medical Research
Laboratory:Mohammad Semreen
Last Name:Hamoudi
First Name:Rifat
Address:College of Medicine, University of Sharjah
Email:rhamoudi@sharjah.ac.ae
Phone:567154756
Funding Source:Al-Jalila Foundation (Grant No: AJF201741), Breast Cancer Now (Grant No: 2014MaySP323) and University of Sharjah
Project Comments:Multidisciplinary based on mass spectrometry and bioinformatics

Subject:

Subject ID:SU001333
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Gender:Female
Cell Biosource Or Supplier:ATCC
Cell Strain Details:MCF-7
Cell Passage Number:30
Species Group:Homo Sapien

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Exctraction
SA092003NH4OH0.2% Ammonium hydroxide in water
SA092004FA0.2% Formic acid in water
SA092005ACN-H2OAcetonitrile/Water
SA092006EthAcetEthyl acetate
SA092007MeOH-H2OMethanol/Water
Showing results 1 to 5 of 5

Collection:

Collection ID:CO001327
Collection Summary:The breast adenocarcinoma cells (MCF-7) were cultured in DMEM medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin solution and incubated at 37oC in an atmosphere of 5% CO2. In preparation for an experiment, 3 × 106 cells were cultured in each of 15 tissue culture flasks (175 cm2) and cells were incubated for three days. When the cells reach 80% confluency, they were collected by trypsinization, counted in a cell counter and each 3 ×106 cells were suspended in 1 ml phosphate-buffered saline in an Eppendorf tube.
Sample Type:Breast cancer cells
Collection Method:trypsinization protocol
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001348
Treatment Summary:The aim of this study was to investigate the solvent extraction efficiency, so there was no cell treatment

Sample Preparation:

Sampleprep ID:SP001341
Sampleprep Summary:A total of 5 triplicated MCF-7 cell culture samples were provided in Eppendorf vials dissolved in PBS, and stored at 4 °C for preservation purposes. Samples were centrifuged at 13000 rpm for 10 minutes at -4 °C. Supernatant was discarded, and cell pellets, each containing 3 million cells, were subjected to metabolomics analysis. In order to evaluate the influence of the solvents on the extraction rate, samples were divided in five different extraction groups: A, 0.2 % formic acid in water; B, 0.2 % ammonium hydroxide in water; C, ethyl acetate; D, methanol/water (1:1, v/v); and E, acetonitrile/water (1:1, v/v). Briefly, 300 µL of the extraction solvent was added to 3 million cell pellets, then vortexed for 2 minutes. All samples have been stored in ice for 1 hour, during which samples have been vortexed every 15 minutes. After this, cell insoluble matrix was centrifuged (13000 rpm, 10 minutes, -4 °C). Supernatant was collected then dried using EZ-2 Plus (GeneVac, Ipswich, UK) at 37 ± 1 °C. To detect all amino acids and metabolites, all samples were derivatized with methoxyamine hydrochloride and MSTFA + 1% TMCS prior to injection to GC-MS.
Processing Storage Conditions:Described in summary
Extraction Method:Direct extraction
Extract Enrichment:described in Summary
Extract Storage:Described in summary
Sample Derivatization:methoxyamine hydrochloride and MSTFA + 1% TMCS

Combined analysis:

Analysis ID AN002102
Analysis type MS
Chromatography type GC
Chromatography system Shimadzu GCMS-QP2010 Ultra
Column Restek Rtx-5Sil MS (30 x 0.25mm, 0.25um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Shimadzu QP2010 Ultra
Ion Mode POSITIVE
Units Peak Area

Chromatography:

Chromatography ID:CH001534
Chromatography Summary:A GC/MS-QP 2010 Ultra System (Shimadzu, Kyoto, Japan) was employed for the metabolomic analysis, along with LabSolutions GC-MS software (ver). A Restek Rtx®-5ms column (30.0 m × 0.25 mm, 0.25 µm) was utilized for separation of the metabolites. Helium (99.9%) was used as the carrier gas at the flow rate of 1.0 mL/min. The initial oven temperature was set at 60 °C and was held at this temperature for 2 minutes, then raised to 310 °C by 50 °C/min and held at this temperature during the analysis. Both the auxiliary temperature at the interface and the ionization temperature were 250 °C. Metabolites were analysed in full scan mode within the range of 50 – 650 amu. Total volume of 10 µL was injected in splitless mode, by employing AOC-20i Auto Injector (Shimadzu, Kyoto, Japan). GC total ion chromatograms (TIC) and fragmentation patterns of the metabolites identified using NIST/EPA/NIH Mass Spectral Library (NIST 14). Run time for each sample was 43.67 min.
Methods Filename:PAH scan
Instrument Name:Shimadzu GCMS-QP2010 Ultra
Column Name:Restek Rtx-5Sil MS (30 x 0.25mm, 0.25um)
Column Pressure:57.4
Column Temperature:60
Flow Rate:1
Injection Temperature:250
Retention Time:many retention time
Sample Injection:1
Solvent A:Pyridine
Analytical Time:43.67
Oven Temperature:60
Time Program:43.67
Transferline Temperature:250
Strong Wash Solvent Name:Acetone
Strong Wash Volume:10 uL
Sample Syringe Size:10 uL
Chromatography Type:GC

MS:

MS ID:MS001953
Analysis ID:AN002102
Instrument Name:Shimadzu QP2010 Ultra
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:A GC/MS-QP 2010 Ultra System (Shimadzu, Kyoto, Japan) was employed for the metabolomic analysis, along with LabSolutions GC-MS software (ver). A Restek Rtx®-5ms column (30.0 m × 0.25 mm, 0.25 µm) was utilized for separation of the metabolites. Helium (99.9%) was used as the carrier gas at the flow rate of 1.0 mL/min. The initial oven temperature was set at 60 °C and was held at this temperature for 2 minutes, then raised to 310 °C by 50 °C/min and held at this temperature during the analysis. Both the auxiliary temperature at the interface and the ionization temperature were 250 °C. Metabolites were analysed in full scan mode within the range of 50 – 650 amu. Total volume of 10 µL was injected in splitless mode, by employing AOC-20i Auto Injector (Shimadzu, Kyoto, Japan). GC total ion chromatograms (TIC) and fragmentation patterns of the metabolites identified using NIST/EPA/NIH Mass Spectral Library (NIST 14). Run time for each sample was 43.67 min.
Ion Mode:POSITIVE
Gas Pressure:57.4 kPa
Helium Flow:1 ml/min
Ion Source Temperature:250
Ionization Energy:70
Source Temperature:250
Scanning Range:50-650
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