Summary of Study ST002543
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 PR001638. The data can be accessed directly via it's Project DOI: 10.21228/M8ZX4D This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST002543 |
| Study Title | GC/MS analysis of hypoxic volatile metabolic markers in the MDA-MB-231 breast cancer cell line |
| Study Summary | Hypoxia in disease describes persistent low oxygen conditions, observed in a range of pathologies, including cancer. In the discovery of biomarkers in biological models, pathophysiological traits present a source of translatable metabolic products for the diagnosis of disease in humans. Part of the metabolome is represented by its volatile, gaseous fraction; the volatilome. Human volatile profiles, such as those found in breath, are able to diagnose disease, however accurate volatile biomarker discovery is required to target reliable biomarkers to develop new diagnostic tools. Using custom chambers to control oxygen levels and facilitate headspace sampling, the MDA-MB-231 breast cancer cell line was exposed to hypoxia (1% oxygen) for 24 hours. The maintenance of hypoxic conditions in the system was successfully validated over this time period. Targeted and untargeted gas chromatography mass spectrometry approaches revealed four significantly altered volatile organic compounds when compared to control cells. Three compounds were actively consumed by cells: methyl chloride, acetone and n-Hexane. Cells under hypoxia also produced significant amounts of styrene. This work presents a novel methodology for identification of volatile metabolisms under controlled gas conditions with novel observations of volatile metabolisms by breast cancer cells. |
| Institute | University of York |
| Last Name | Issitt |
| First Name | Theo |
| Address | Biology Dept. University of York, Personal |
| ti538@york.ac.uk | |
| Phone | 07398244497 |
| Submit Date | 2023-03-31 |
| Num Groups | 4 |
| Publications | T. Issitt et al., Volatile compounds in human breath: critical review and meta-analysis Journal of Breath Research, Volume 16, Number 2 (2022) https://iopscience.iop.org/article/10.1088/1752-7163/ac5230#jbrac5230s2 |
| Analysis Type Detail | GC-MS |
| Release Date | 2023-04-21 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001638 |
| Project DOI: | doi: 10.21228/M8ZX4D |
| Project Title: | GC/MS analysis of hypoxic volatile metabolic markers in the MDA-MB-231 breast cancer cell line |
| Project Summary: | Hypoxia in disease describes persistent low oxygen conditions, observed in a range of pathologies, including cancer. In the discovery of biomarkers in biological models, pathophysiological traits present a source of translatable metabolic products for the diagnosis of disease in humans. Part of the metabolome is represented by its volatile, gaseous fraction; the volatilome. Human volatile profiles, such as those found in breath, are able to diagnose disease, however accurate volatile biomarker discovery is required to target reliable biomarkers to develop new diagnostic tools. Using custom chambers to control oxygen levels and facilitate headspace sampling, the MDA-MB-231 breast cancer cell line was exposed to hypoxia (1% oxygen) for 24 hours. The maintenance of hypoxic conditions in the system was successfully validated over this time period. Targeted and ununtargeted gas chromatography mass spectrometry approaches revealed four significantly altered volatile organic compounds when compared to control cells. Three compounds were actively consumed by cells: methyl chloride, acetone and n-Hexane. Cells under hypoxia also produced significant amounts of styrene. This work presents a novel methodology for identification of volatile metabolisms under controlled gas conditions with novel observations of volatile metabolisms by breast cancer cells. |
| Institute: | University of York |
| Department: | Biology |
| Last Name: | Issitt |
| First Name: | Theo |
| Address: | Biology Dept. University of York |
| Email: | ti538@york.ac.uk |
| Phone: | 07398244497 |
| Funding Source: | BBSRC |
| Publications: | T. Issitt et al., Volatile compounds in human breath: critical review and meta-analysis Journal of Breath Research, Volume 16, Number 2 (2022) https://iopscience.iop.org/article/10.1088/1752-7163/ac5230#jbrac5230s2 |
Subject:
| Subject ID: | SU002643 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Strain Details: | MDA-MB-231 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample Type | Treatment |
|---|---|---|---|
| SA255602 | c3 | Cells | Control |
| SA255603 | c4 | Cells | Control |
| SA255604 | c6 | Cells | Control |
| SA255605 | c2 | Cells | Control |
| SA255606 | c5 | Cells | Control |
| SA255607 | c1 | Cells | Control |
| SA255608 | h2 | Cells | Hypoxia |
| SA255609 | h1 | Cells | Hypoxia |
| SA255610 | h3 | Cells | Hypoxia |
| SA255611 | h4 | Cells | Hypoxia |
| SA255612 | h5 | Cells | Hypoxia |
| SA255613 | h6 | Cells | Hypoxia |
| SA255614 | m3 | Media | Control |
| SA255615 | m2 | Media | Control |
| SA255616 | m4 | Media | Control |
| SA255617 | m5 | Media | Control |
| SA255618 | m6 | Media | Control |
| SA255619 | m1 | Media | Control |
| SA255620 | hm4 | Media | Hypoxia |
| SA255621 | hm3 | Media | Hypoxia |
| SA255622 | hm5 | Media | Hypoxia |
| SA255623 | hm6 | Media | Hypoxia |
| SA255624 | hm2 | Media | Hypoxia |
| SA255625 | hm1 | Media | Hypoxia |
| Showing results 1 to 24 of 24 |
Collection:
| Collection ID: | CO002636 |
| Collection Summary: | Cells were placed in static headspace chambers as previously described [4] with new, clean silicon gaskets. Low oxygen, hypoxic gas (1 % O2, 5 % CO2, 94 % N2; purchased from BOC Specialty Gases, Woking, UK) was flushed through the chambers at a rate of 4 L/min for 10 min (chamber volume = 25 L). Chambers were then closed and placed at 37 ˚C for 2 hours to allow residual oxygen in the media to equilibrate with chamber headspace. Chambers were then flushed again at a rate of 4 L/min for 10 min, sealed and returned to 37 ˚C. After a further 24 hours, chambers were flushed again at a rate of 4 L/min for 10 min. 15 ml of gas standards (MeCl, 520 ppb (parts per billion); MeBr, 22 ppb; MeI, 26 ppb; DMS, 110 ppb; CFC-11, 400 ppb and CH3Cl3, 110ppb; BOC Specialty Gases, Woking, UK) were then injected into the chambers through a butyl seal and time zero sample taken. Injected compounds are either known metabolites for cancer cells, or internal standards (CFC-11) for the analysis and quantification of metabolism. Final chamber concentrations were similar to environmental concentrations, e.g MeCl, 1.2 ppb and MeBr 0.05 ppb, particularly more polluted urban spaces (Redeker et al., 2007). Injected gases are the same as those used for calibration. Compounds not injected but detected at first time point, due to residual presence from laboratory air, (including isoprene, acetone, 2-MP, 3-MP and n-hexane) were quantified. Two time zero (T0) samples were taken using an evacuated 500 mL electropolished stainless steel canister (LabCommerce, San Jose, USA) through fine mesh Ascarite® traps (Archbold et al., 2005), after which the chamber was resealed and left on a platform rocker on its slowest setting for 120 min, at which point two further air samples (T1) were collected. Duplicate samples were taken so that two analytical approaches could be performed (targeted and non-untargeted MS). Cells were removed from the chamber, washed with PBS twice and lysed in 500 µL RIPA buffer (NaCl (5 M), 5 mL Tris-HCl (1 M, pH 8.0), 1 mL Nonidet P-40, 5 mL sodium deoxycholate (10 %), 1 mL SDS (10 %)) with protease inhibitor (Sigma-Aldrich, Roche; Mannheim, Germany). Protein concentration of lysates were determined using BCA assay (Thermo Scientific, Waltham, MA, USA). Media alone was treated exactly the same as cells, and only acetone was found to differ significantly between conditions (Supplementary figure 1). These media blank outcomes were subtracted from respective cellular samples prior to protein normalisation. Comparative controls include lab air blanks and those data available from the dataset and collection method published previously which created and quantified metabolic fluxes of volatile compounds from MDA-MB-231 under hyperoxic (lab air) conditions (Issitt et al., 2022a). |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR002655 |
| Treatment Summary: | Cells were placed in static headspace chambers as previously described [4] with new, clean silicon gaskets. Low oxygen, hypoxic gas (1 % O2, 5 % CO2, 94 % N2; purchased from BOC Specialty Gases, Woking, UK) was flushed through the chambers at a rate of 4 L/min for 10 min (chamber volume = 25 L). Chambers were then closed and placed at 37 ˚C for 2 hours to allow residual oxygen in the media to equilibrate with chamber headspace. Chambers were then flushed again at a rate of 4 L/min for 10 min, sealed and returned to 37 ˚C. After a further 24 hours, chambers were flushed again at a rate of 4 L/min for 10 min. 15 ml of gas standards (MeCl, 520 ppb (parts per billion); MeBr, 22 ppb; MeI, 26 ppb; DMS, 110 ppb; CFC-11, 400 ppb and CH3Cl3, 110ppb; BOC Specialty Gases, Woking, UK) were then injected into the chambers through a butyl seal and time zero sample taken. Injected compounds are either known metabolites for cancer cells, or internal standards (CFC-11) for the analysis and quantification of metabolism. Final chamber concentrations were similar to environmental concentrations, e.g MeCl, 1.2 ppb and MeBr 0.05 ppb, particularly more polluted urban spaces (Redeker et al., 2007). Injected gases are the same as those used for calibration. Compounds not injected but detected at first time point, due to residual presence from laboratory air, (including isoprene, acetone, 2-MP, 3-MP and n-hexane) were quantified. Two time zero (T0) samples were taken using an evacuated 500 mL electropolished stainless steel canister (LabCommerce, San Jose, USA) through fine mesh Ascarite® traps (Archbold et al., 2005), after which the chamber was resealed and left on a platform rocker on its slowest setting for 120 min, at which point two further air samples (T1) were collected. Duplicate samples were taken so that two analytical approaches could be performed (targeted and non-untargeted MS). Cells were removed from the chamber, washed with PBS twice and lysed in 500 µL RIPA buffer (NaCl (5 M), 5 mL Tris-HCl (1 M, pH 8.0), 1 mL Nonidet P-40, 5 mL sodium deoxycholate (10 %), 1 mL SDS (10 %)) with protease inhibitor (Sigma-Aldrich, Roche; Mannheim, Germany). Protein concentration of lysates were determined using BCA assay (Thermo Scientific, Waltham, MA, USA). Media alone was treated exactly the same as cells, and only acetone was found to differ significantly between conditions (Supplementary figure 1). These media blank outcomes were subtracted from respective cellular samples prior to protein normalisation. Comparative controls include lab air blanks and those data available from the dataset and collection method published previously which created and quantified metabolic fluxes of volatile compounds from MDA-MB-231 under hyperoxic (lab air) conditions (Issitt et al., 2022a). |
| Treatment: | Hypoxia |
| Treatment Vehicle: | Nitrogen |
| Cell Storage: | 37 degrees |
| Cell Media: | DMEM |
| Cell Envir Cond: | Hypoxia/lab air |
Sample Preparation:
| Sampleprep ID: | SP002649 |
| Sampleprep Summary: | Collected canister samples were transferred to a liquid nitrogen trap through pressure differential. Pressure change between beginning and end of “injection” was measured, allowing calculation of the moles of canister collected air injected. Sample in the trap was then transferred, via heated helium flow, to an Aglient/HP 5972 MSD system (Santa Clara, CA, USA) equipped with a PoraBond Q column (25 m x 0.32 mm x 0.5 μm film thickness) (Restek©, Bellefonte, PN, USA). Targeted samples were analyzed in selected ion monitoring (SIM) mode, and untargeted samples in full scan (SCAN) mode with the mass range of 45-200 amu. The mass spectrometer was operated in electron impact ionization mode with 70 eV ionization energy, and transfer line, ion source, and quadrupole temperatures of 250, 280 and 280, respectively. For details on SIM and significantly altered, identified SCAN compounds, see Table 1. All samples were analysed within 6 days of collection. The oven program for both SIM and SCAN analyses were identical and are as follows: 35 ˚C for 2 min, 10 ˚C/min to 155 ˚C, 1 ˚C/min to 131 ˚C and 25 ˚C/min to 250 with a 5 min 30 second hold. Calibration was performed using standard gases (BOC Specialty Gases, Woking, UK). Linear regression of calibration curves confirmed strong, positive linear relationships between observed compound peak areas and moles of gas injected for each VOC (r2 > 0.9 in all cases). For compounds not purchased in gaseous state (BOC Specialty gases, as above), 1–2 mL of compound in liquid phase was injected neat into butyl sealed Wheaton-style glass vials (100 mL) and allowed to equilibrate for 1 h. 1 mL of headspace air was then removed from neat vial headspace using a gas tight syringe (Trajan, SGE) and injected into the headspace of a second 100 mL butyl sealed Wheaton-style glass vial. This was then repeated, and 1 mL of the 2nd serial dilution vial was injected into the GCMS system with 29 mL of lab air to give ppb concentrations. This was performed for methanethiol (MeSH (SPEXorganics, St Neots, UK)), isoprene (Alfa Aesar, Ward Hill, MA, USA), acetone (Sigma-Aldrich, Burlington, MA, USA), 2- & 3-methyl pentane and n-hexane (Thermo Scientific, Waltham, MA, USA). Reported compounds detected by the GC/-MS were confirmed by matching retention times and mass–charge (m/z) ratios with known standards. |
| Processing Storage Conditions: | Room temperature |
Combined analysis:
| Analysis ID | AN004190 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | HP GCD 1800B |
| Column | Agilent PoraBOND Q (25m x 0.32mm x 0.5um) |
| MS Type | EI |
| MS instrument type | Single quadrupole |
| MS instrument name | Agilent/HP 5972 MSD |
| Ion Mode | POSITIVE |
| Units | pg/hr/ug and g/hr for media |
Chromatography:
| Chromatography ID: | CH003104 |
| Chromatography Summary: | Collected canister samples were transferred to a liquid nitrogen trap through pressure differential. Pressure change between beginning and end of “injection” was measured, allowing calculation of the moles of canister collected air injected. Sample in the trap was then transferred, via heated helium flow, to an Aglient/HP 5972 MSD system (Santa Clara, CA, USA) equipped with a PoraBond Q column (25 m x 0.32 mm x 0.5 μm film thickness) (Restek©, Bellefonte, PN, USA). Targeted samples were analyzed in selected ion monitoring (SIM) mode, and untargeted samples in full scan (SCAN) mode with the mass range of 45-200 amu. The mass spectrometer was operated in electron impact ionization mode with 70 eV ionization energy, and transfer line, ion source, and quadrupole temperatures of 250, 280 and 280, respectively. For details on SIM and significantly altered, identified SCAN compounds, see Table 1. All samples were analysed within 6 days of collection. The oven program for both SIM and SCAN analyses were identical and are as follows: 35 ˚C for 2 min, 10 ˚C/min to 155 ˚C, 1 ˚C/min to 131 ˚C and 25 ˚C/min to 250 with a 5 min 30 second hold. |
| Instrument Name: | HP GCD 1800B |
| Column Name: | Agilent PoraBOND Q (25m x 0.32mm x 0.5um) |
| Column Temperature: | 250 |
| Flow Gradient: | NA |
| Flow Rate: | 10ml/min |
| Solvent A: | NA |
| Solvent B: | NA |
| Chromatography Type: | GC |
MS:
| MS ID: | MS003937 |
| Analysis ID: | AN004190 |
| Instrument Name: | Agilent/HP 5972 MSD |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | Calibration was performed using standard gases (BOC Specialty Gases, Woking, UK). Linear regression of calibration curves confirmed strong, positive linear relationships between observed compound peak areas and moles of gas injected for each VOC (r2 > 0.9 in all cases). For compounds not purchased in gaseous state (BOC Specialty gases, as above), 1–2 mL of compound in liquid phase was injected neat into butyl sealed Wheaton-style glass vials (100 mL) and allowed to equilibrate for 1 h. 1 mL of headspace air was then removed from neat vial headspace using a gas tight syringe (Trajan, SGE) and injected into the headspace of a second 100 mL butyl sealed Wheaton-style glass vial. This was then repeated, and 1 mL of the 2nd serial dilution vial was injected into the GCMS system with 29 mL of lab air to give ppb concentrations. This was performed for methanethiol (MeSH (SPEXorganics, St Neots, UK)), isoprene (Alfa Aesar, Ward Hill, MA, USA), acetone (Sigma-Aldrich, Burlington, MA, USA), 2- & 3-methyl pentane and n-hexane (Thermo Scientific, Waltham, MA, USA). Reported compounds detected by the GC/-MS were confirmed by matching retention times and mass–charge (m/z) ratios with known standards. Equation 1: [VOC](ppt)=(CF x 〖10〗^12 x Peak area x Calibration slope)/n Equation 1 outlines the approach to calculating VOC concentrations in parts-per-trillion-by-volume, or pptv. Here Peak area refers to the combined peak areas for the mass-charge ratios identified in Table 1. Multiplying Peak areas by their associated calibration curves (Calibration Slope) generate molar amounts which, when divided by the number of moles of headspace air injected (n), generate a unitless (moles compound/moles of air) ratio. Pptv concentrations are then obtained by multiplying this unitless ratio by 1x1012. For clarity, part-per-billion-by-volume values would be obtained by multiplying the unitless ratios by 1x109, or one billion. Sample VOC concentrations were then normalised to CFC-11 concentrations (240 parts-per-trillion-by-volume (pptv)) through multiplication by a “correction factor”, or CF, Equation 1). CFC-11 was used as an internal standard, since atmospheric concentrations of CFC-11 are globally consistent and stable (Redeker et al., 2007). Quantification of Styrene was done as above but normalisation to CFC-11 was not possible under flushed, hypoxic conditions. NEGATIVE VALUES IN DATA SHOW CONSUMPTION OVER TIME. VARIATION IN SCALE BETWEEN MEDIA SAMPLES ARE DUE TO NORMALISATION OF CELLULAR DATA TO PROTEIN. AS DESCRIBED, MEDIA VALUES ARE SUBTRACTED FROM CELLULAR DATA PRIOR TO NORMALISATION AND EXPRESSED AS PG/HR/UG. |
| Ion Mode: | POSITIVE |
