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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001265 |
| Study Title | Comparative metabolomics of MCF-7 breast cancer cells using different extraction solvents assessed by mass spectroscopy |
| Study Type | Analysing metabolomics using GC Mass Spectroscopy |
| Study Summary | Metabolic 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 |
| Department | Clinical Science |
| Last Name | Hamoudi |
| First Name | Rifat |
| Address | College of Medicine, University of Sharjah |
| rhamoudi@sharjah.ac.ae | |
| Phone | 567154756 |
| Submit Date | 2019-07-08 |
| Total Subjects | Five different extractions |
| Study Comments | MCF-7 cell line |
| Raw Data Available | Yes |
| Raw Data File Type(s) | gqd |
| Analysis Type Detail | GC-MS |
| Release Date | 2019-10-11 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
|---|---|---|
| SA092003 | NH4OH | 0.2% Ammonium hydroxide in water |
| SA092004 | FA | 0.2% Formic acid in water |
| SA092005 | ACN-H2O | Acetonitrile/Water |
| SA092006 | EthAcet | Ethyl acetate |
| SA092007 | MeOH-H2O | Methanol/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 (30m 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 (30m 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 |
