#METABOLOMICS WORKBENCH theoissitt_20230331_063813 DATATRACK_ID:3830 STUDY_ID:ST002543 ANALYSIS_ID:AN004190 PROJECT_ID:PR001638 VERSION 1 CREATED_ON April 6, 2023, 3:07 pm #PROJECT PR:PROJECT_TITLE GC/MS analysis of hypoxic volatile metabolic markers in the MDA-MB-231 breast PR:PROJECT_TITLE cancer cell line PR:PROJECT_SUMMARY Hypoxia in disease describes persistent low oxygen conditions, observed in a PR:PROJECT_SUMMARY range of pathologies, including cancer. In the discovery of biomarkers in PR:PROJECT_SUMMARY biological models, pathophysiological traits present a source of translatable PR:PROJECT_SUMMARY metabolic products for the diagnosis of disease in humans. Part of the PR:PROJECT_SUMMARY metabolome is represented by its volatile, gaseous fraction; the volatilome. PR:PROJECT_SUMMARY Human volatile profiles, such as those found in breath, are able to diagnose PR:PROJECT_SUMMARY disease, however accurate volatile biomarker discovery is required to target PR:PROJECT_SUMMARY reliable biomarkers to develop new diagnostic tools. Using custom chambers to PR:PROJECT_SUMMARY control oxygen levels and facilitate headspace sampling, the MDA-MB-231 breast PR:PROJECT_SUMMARY cancer cell line was exposed to hypoxia (1% oxygen) for 24 hours. The PR:PROJECT_SUMMARY maintenance of hypoxic conditions in the system was successfully validated over PR:PROJECT_SUMMARY this time period. Targeted and ununtargeted gas chromatography mass spectrometry PR:PROJECT_SUMMARY approaches revealed four significantly altered volatile organic compounds when PR:PROJECT_SUMMARY compared to control cells. Three compounds were actively consumed by cells: PR:PROJECT_SUMMARY methyl chloride, acetone and n-Hexane. Cells under hypoxia also produced PR:PROJECT_SUMMARY significant amounts of styrene. This work presents a novel methodology for PR:PROJECT_SUMMARY identification of volatile metabolisms under controlled gas conditions with PR:PROJECT_SUMMARY novel observations of volatile metabolisms by breast cancer cells. PR:INSTITUTE University of York PR:DEPARTMENT Biology PR:LAST_NAME Issitt PR:FIRST_NAME Theo PR:ADDRESS Biology Dept. University of York PR:EMAIL ti538@york.ac.uk PR:PHONE 07398244497 PR:FUNDING_SOURCE BBSRC PR:PUBLICATIONS T. Issitt et al., Volatile compounds in human breath: critical review and PR:PUBLICATIONS meta-analysis Journal of Breath Research, Volume 16, Number 2 (2022) PR:PUBLICATIONS https://iopscience.iop.org/article/10.1088/1752-7163/ac5230#jbrac5230s2 #STUDY ST:STUDY_TITLE GC/MS analysis of hypoxic volatile metabolic markers in the MDA-MB-231 breast ST:STUDY_TITLE cancer cell line ST:STUDY_SUMMARY Hypoxia in disease describes persistent low oxygen conditions, observed in a ST:STUDY_SUMMARY range of pathologies, including cancer. In the discovery of biomarkers in ST:STUDY_SUMMARY biological models, pathophysiological traits present a source of translatable ST:STUDY_SUMMARY metabolic products for the diagnosis of disease in humans. Part of the ST:STUDY_SUMMARY metabolome is represented by its volatile, gaseous fraction; the volatilome. ST:STUDY_SUMMARY Human volatile profiles, such as those found in breath, are able to diagnose ST:STUDY_SUMMARY disease, however accurate volatile biomarker discovery is required to target ST:STUDY_SUMMARY reliable biomarkers to develop new diagnostic tools. Using custom chambers to ST:STUDY_SUMMARY control oxygen levels and facilitate headspace sampling, the MDA-MB-231 breast ST:STUDY_SUMMARY cancer cell line was exposed to hypoxia (1% oxygen) for 24 hours. The ST:STUDY_SUMMARY maintenance of hypoxic conditions in the system was successfully validated over ST:STUDY_SUMMARY this time period. Targeted and untargeted gas chromatography mass spectrometry ST:STUDY_SUMMARY approaches revealed four significantly altered volatile organic compounds when ST:STUDY_SUMMARY compared to control cells. Three compounds were actively consumed by cells: ST:STUDY_SUMMARY methyl chloride, acetone and n-Hexane. Cells under hypoxia also produced ST:STUDY_SUMMARY significant amounts of styrene. This work presents a novel methodology for ST:STUDY_SUMMARY identification of volatile metabolisms under controlled gas conditions with ST:STUDY_SUMMARY novel observations of volatile metabolisms by breast cancer cells. ST:INSTITUTE University of York ST:LAST_NAME Issitt ST:FIRST_NAME Theo ST:ADDRESS Biology Dept. University of York, Personal ST:EMAIL ti538@york.ac.uk ST:NUM_GROUPS 4 ST:PUBLICATIONS T. Issitt et al., Volatile compounds in human breath: critical review and ST:PUBLICATIONS meta-analysis Journal of Breath Research, Volume 16, Number 2 (2022) ST:PUBLICATIONS https://iopscience.iop.org/article/10.1088/1752-7163/ac5230#jbrac5230s2 ST:PHONE 07398244497 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS MDA-MB-231 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - m1 Sample Type:Media | Treatment:Control SUBJECT_SAMPLE_FACTORS - m2 Sample Type:Media | Treatment:Control SUBJECT_SAMPLE_FACTORS - m3 Sample Type:Media | Treatment:Control SUBJECT_SAMPLE_FACTORS - m4 Sample Type:Media | Treatment:Control SUBJECT_SAMPLE_FACTORS - m5 Sample Type:Media | Treatment:Control SUBJECT_SAMPLE_FACTORS - m6 Sample Type:Media | Treatment:Control SUBJECT_SAMPLE_FACTORS - hm1 Sample Type:Media | Treatment:Hypoxia SUBJECT_SAMPLE_FACTORS - hm2 Sample Type:Media | Treatment:Hypoxia SUBJECT_SAMPLE_FACTORS - hm3 Sample Type:Media | Treatment:Hypoxia SUBJECT_SAMPLE_FACTORS - hm4 Sample Type:Media | Treatment:Hypoxia SUBJECT_SAMPLE_FACTORS - hm5 Sample Type:Media | Treatment:Hypoxia SUBJECT_SAMPLE_FACTORS - hm6 Sample Type:Media | Treatment:Hypoxia SUBJECT_SAMPLE_FACTORS - c1 Sample Type:Cells | Treatment:Control SUBJECT_SAMPLE_FACTORS - c2 Sample Type:Cells | Treatment:Control SUBJECT_SAMPLE_FACTORS - c3 Sample Type:Cells | Treatment:Control SUBJECT_SAMPLE_FACTORS - c4 Sample Type:Cells | Treatment:Control SUBJECT_SAMPLE_FACTORS - c5 Sample Type:Cells | Treatment:Control SUBJECT_SAMPLE_FACTORS - c6 Sample Type:Cells | Treatment:Control SUBJECT_SAMPLE_FACTORS - h1 Sample Type:Cells | Treatment:Hypoxia SUBJECT_SAMPLE_FACTORS - h2 Sample Type:Cells | Treatment:Hypoxia SUBJECT_SAMPLE_FACTORS - h3 Sample Type:Cells | Treatment:Hypoxia SUBJECT_SAMPLE_FACTORS - h4 Sample Type:Cells | Treatment:Hypoxia SUBJECT_SAMPLE_FACTORS - h5 Sample Type:Cells | Treatment:Hypoxia SUBJECT_SAMPLE_FACTORS - h6 Sample Type:Cells | Treatment:Hypoxia #COLLECTION CO:COLLECTION_SUMMARY Cells were placed in static headspace chambers as previously described [4] with CO:COLLECTION_SUMMARY new, clean silicon gaskets. Low oxygen, hypoxic gas (1 % O2, 5 % CO2, 94 % N2; CO:COLLECTION_SUMMARY purchased from BOC Specialty Gases, Woking, UK) was flushed through the chambers CO:COLLECTION_SUMMARY at a rate of 4 L/min for 10 min (chamber volume = 25 L). Chambers were then CO:COLLECTION_SUMMARY closed and placed at 37 ˚C for 2 hours to allow residual oxygen in the media to CO:COLLECTION_SUMMARY equilibrate with chamber headspace. Chambers were then flushed again at a rate CO:COLLECTION_SUMMARY of 4 L/min for 10 min, sealed and returned to 37 ˚C. After a further 24 hours, CO:COLLECTION_SUMMARY chambers were flushed again at a rate of 4 L/min for 10 min. 15 ml of gas CO:COLLECTION_SUMMARY standards (MeCl, 520 ppb (parts per billion); MeBr, 22 ppb; MeI, 26 ppb; DMS, CO:COLLECTION_SUMMARY 110 ppb; CFC-11, 400 ppb and CH3Cl3, 110ppb; BOC Specialty Gases, Woking, UK) CO:COLLECTION_SUMMARY were then injected into the chambers through a butyl seal and time zero sample CO:COLLECTION_SUMMARY taken. Injected compounds are either known metabolites for cancer cells, or CO:COLLECTION_SUMMARY internal standards (CFC-11) for the analysis and quantification of metabolism. CO:COLLECTION_SUMMARY Final chamber concentrations were similar to environmental concentrations, e.g CO:COLLECTION_SUMMARY MeCl, 1.2 ppb and MeBr 0.05 ppb, particularly more polluted urban spaces CO:COLLECTION_SUMMARY (Redeker et al., 2007). Injected gases are the same as those used for CO:COLLECTION_SUMMARY calibration. Compounds not injected but detected at first time point, due to CO:COLLECTION_SUMMARY residual presence from laboratory air, (including isoprene, acetone, 2-MP, 3-MP CO:COLLECTION_SUMMARY and n-hexane) were quantified. Two time zero (T0) samples were taken using an CO:COLLECTION_SUMMARY evacuated 500 mL electropolished stainless steel canister (LabCommerce, San CO:COLLECTION_SUMMARY Jose, USA) through fine mesh Ascarite® traps (Archbold et al., 2005), after CO:COLLECTION_SUMMARY which the chamber was resealed and left on a platform rocker on its slowest CO:COLLECTION_SUMMARY setting for 120 min, at which point two further air samples (T1) were collected. CO:COLLECTION_SUMMARY Duplicate samples were taken so that two analytical approaches could be CO:COLLECTION_SUMMARY performed (targeted and non-untargeted MS). Cells were removed from the chamber, CO:COLLECTION_SUMMARY washed with PBS twice and lysed in 500 µL RIPA buffer (NaCl (5 M), 5 mL CO:COLLECTION_SUMMARY Tris-HCl (1 M, pH 8.0), 1 mL Nonidet P-40, 5 mL sodium deoxycholate (10 %), 1 mL CO:COLLECTION_SUMMARY SDS (10 %)) with protease inhibitor (Sigma-Aldrich, Roche; Mannheim, Germany). CO:COLLECTION_SUMMARY Protein concentration of lysates were determined using BCA assay (Thermo CO:COLLECTION_SUMMARY Scientific, Waltham, MA, USA). Media alone was treated exactly the same as CO:COLLECTION_SUMMARY cells, and only acetone was found to differ significantly between conditions CO:COLLECTION_SUMMARY (Supplementary figure 1). These media blank outcomes were subtracted from CO:COLLECTION_SUMMARY respective cellular samples prior to protein normalisation. Comparative controls CO:COLLECTION_SUMMARY include lab air blanks and those data available from the dataset and collection CO:COLLECTION_SUMMARY method published previously which created and quantified metabolic fluxes of CO:COLLECTION_SUMMARY volatile compounds from MDA-MB-231 under hyperoxic (lab air) conditions (Issitt CO:COLLECTION_SUMMARY et al., 2022a). CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Cells were placed in static headspace chambers as previously described [4] with TR:TREATMENT_SUMMARY new, clean silicon gaskets. Low oxygen, hypoxic gas (1 % O2, 5 % CO2, 94 % N2; TR:TREATMENT_SUMMARY purchased from BOC Specialty Gases, Woking, UK) was flushed through the chambers TR:TREATMENT_SUMMARY at a rate of 4 L/min for 10 min (chamber volume = 25 L). Chambers were then TR:TREATMENT_SUMMARY closed and placed at 37 ˚C for 2 hours to allow residual oxygen in the media to TR:TREATMENT_SUMMARY equilibrate with chamber headspace. Chambers were then flushed again at a rate TR:TREATMENT_SUMMARY of 4 L/min for 10 min, sealed and returned to 37 ˚C. After a further 24 hours, TR:TREATMENT_SUMMARY chambers were flushed again at a rate of 4 L/min for 10 min. 15 ml of gas TR:TREATMENT_SUMMARY standards (MeCl, 520 ppb (parts per billion); MeBr, 22 ppb; MeI, 26 ppb; DMS, TR:TREATMENT_SUMMARY 110 ppb; CFC-11, 400 ppb and CH3Cl3, 110ppb; BOC Specialty Gases, Woking, UK) TR:TREATMENT_SUMMARY were then injected into the chambers through a butyl seal and time zero sample TR:TREATMENT_SUMMARY taken. Injected compounds are either known metabolites for cancer cells, or TR:TREATMENT_SUMMARY internal standards (CFC-11) for the analysis and quantification of metabolism. TR:TREATMENT_SUMMARY Final chamber concentrations were similar to environmental concentrations, e.g TR:TREATMENT_SUMMARY MeCl, 1.2 ppb and MeBr 0.05 ppb, particularly more polluted urban spaces TR:TREATMENT_SUMMARY (Redeker et al., 2007). Injected gases are the same as those used for TR:TREATMENT_SUMMARY calibration. Compounds not injected but detected at first time point, due to TR:TREATMENT_SUMMARY residual presence from laboratory air, (including isoprene, acetone, 2-MP, 3-MP TR:TREATMENT_SUMMARY and n-hexane) were quantified. Two time zero (T0) samples were taken using an TR:TREATMENT_SUMMARY evacuated 500 mL electropolished stainless steel canister (LabCommerce, San TR:TREATMENT_SUMMARY Jose, USA) through fine mesh Ascarite® traps (Archbold et al., 2005), after TR:TREATMENT_SUMMARY which the chamber was resealed and left on a platform rocker on its slowest TR:TREATMENT_SUMMARY setting for 120 min, at which point two further air samples (T1) were collected. TR:TREATMENT_SUMMARY Duplicate samples were taken so that two analytical approaches could be TR:TREATMENT_SUMMARY performed (targeted and non-untargeted MS). Cells were removed from the chamber, TR:TREATMENT_SUMMARY washed with PBS twice and lysed in 500 µL RIPA buffer (NaCl (5 M), 5 mL TR:TREATMENT_SUMMARY Tris-HCl (1 M, pH 8.0), 1 mL Nonidet P-40, 5 mL sodium deoxycholate (10 %), 1 mL TR:TREATMENT_SUMMARY SDS (10 %)) with protease inhibitor (Sigma-Aldrich, Roche; Mannheim, Germany). TR:TREATMENT_SUMMARY Protein concentration of lysates were determined using BCA assay (Thermo TR:TREATMENT_SUMMARY Scientific, Waltham, MA, USA). Media alone was treated exactly the same as TR:TREATMENT_SUMMARY cells, and only acetone was found to differ significantly between conditions TR:TREATMENT_SUMMARY (Supplementary figure 1). These media blank outcomes were subtracted from TR:TREATMENT_SUMMARY respective cellular samples prior to protein normalisation. Comparative controls TR:TREATMENT_SUMMARY include lab air blanks and those data available from the dataset and collection TR:TREATMENT_SUMMARY method published previously which created and quantified metabolic fluxes of TR:TREATMENT_SUMMARY volatile compounds from MDA-MB-231 under hyperoxic (lab air) conditions (Issitt TR:TREATMENT_SUMMARY et al., 2022a). TR:TREATMENT Hypoxia TR:TREATMENT_VEHICLE Nitrogen TR:CELL_STORAGE 37 degrees TR:CELL_MEDIA DMEM TR:CELL_ENVIR_COND Hypoxia/lab air #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Collected canister samples were transferred to a liquid nitrogen trap through SP:SAMPLEPREP_SUMMARY pressure differential. Pressure change between beginning and end of SP:SAMPLEPREP_SUMMARY “injection” was measured, allowing calculation of the moles of canister SP:SAMPLEPREP_SUMMARY collected air injected. Sample in the trap was then transferred, via heated SP:SAMPLEPREP_SUMMARY helium flow, to an Aglient/HP 5972 MSD system (Santa Clara, CA, USA) equipped SP:SAMPLEPREP_SUMMARY with a PoraBond Q column (25 m x 0.32 mm x 0.5 μm film thickness) (Restek©, SP:SAMPLEPREP_SUMMARY Bellefonte, PN, USA). Targeted samples were analyzed in selected ion monitoring SP:SAMPLEPREP_SUMMARY (SIM) mode, and untargeted samples in full scan (SCAN) mode with the mass range SP:SAMPLEPREP_SUMMARY of 45-200 amu. The mass spectrometer was operated in electron impact ionization SP:SAMPLEPREP_SUMMARY mode with 70 eV ionization energy, and transfer line, ion source, and quadrupole SP:SAMPLEPREP_SUMMARY temperatures of 250, 280 and 280, respectively. For details on SIM and SP:SAMPLEPREP_SUMMARY significantly altered, identified SCAN compounds, see Table 1. All samples were SP:SAMPLEPREP_SUMMARY analysed within 6 days of collection. The oven program for both SIM and SCAN SP:SAMPLEPREP_SUMMARY analyses were identical and are as follows: 35 ˚C for 2 min, 10 ˚C/min to 155 SP:SAMPLEPREP_SUMMARY ˚C, 1 ˚C/min to 131 ˚C and 25 ˚C/min to 250 with a 5 min 30 second hold. SP:SAMPLEPREP_SUMMARY Calibration was performed using standard gases (BOC Specialty Gases, Woking, SP:SAMPLEPREP_SUMMARY UK). Linear regression of calibration curves confirmed strong, positive linear SP:SAMPLEPREP_SUMMARY relationships between observed compound peak areas and moles of gas injected for SP:SAMPLEPREP_SUMMARY each VOC (r2 > 0.9 in all cases). For compounds not purchased in gaseous state SP:SAMPLEPREP_SUMMARY (BOC Specialty gases, as above), 1–2 mL of compound in liquid phase was SP:SAMPLEPREP_SUMMARY injected neat into butyl sealed Wheaton-style glass vials (100 mL) and allowed SP:SAMPLEPREP_SUMMARY to equilibrate for 1 h. 1 mL of headspace air was then removed from neat vial SP:SAMPLEPREP_SUMMARY headspace using a gas tight syringe (Trajan, SGE) and injected into the SP:SAMPLEPREP_SUMMARY headspace of a second 100 mL butyl sealed Wheaton-style glass vial. This was SP:SAMPLEPREP_SUMMARY then repeated, and 1 mL of the 2nd serial dilution vial was injected into the SP:SAMPLEPREP_SUMMARY GCMS system with 29 mL of lab air to give ppb concentrations. This was performed SP:SAMPLEPREP_SUMMARY for methanethiol (MeSH (SPEXorganics, St Neots, UK)), isoprene (Alfa Aesar, Ward SP:SAMPLEPREP_SUMMARY Hill, MA, USA), acetone (Sigma-Aldrich, Burlington, MA, USA), 2- & 3-methyl SP:SAMPLEPREP_SUMMARY pentane and n-hexane (Thermo Scientific, Waltham, MA, USA). Reported compounds SP:SAMPLEPREP_SUMMARY detected by the GC/-MS were confirmed by matching retention times and SP:SAMPLEPREP_SUMMARY mass–charge (m/z) ratios with known standards. SP:PROCESSING_STORAGE_CONDITIONS Room temperature #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Collected canister samples were transferred to a liquid nitrogen trap through CH:CHROMATOGRAPHY_SUMMARY pressure differential. Pressure change between beginning and end of CH:CHROMATOGRAPHY_SUMMARY “injection” was measured, allowing calculation of the moles of canister CH:CHROMATOGRAPHY_SUMMARY collected air injected. Sample in the trap was then transferred, via heated CH:CHROMATOGRAPHY_SUMMARY helium flow, to an Aglient/HP 5972 MSD system (Santa Clara, CA, USA) equipped CH:CHROMATOGRAPHY_SUMMARY with a PoraBond Q column (25 m x 0.32 mm x 0.5 μm film thickness) (Restek©, CH:CHROMATOGRAPHY_SUMMARY Bellefonte, PN, USA). Targeted samples were analyzed in selected ion monitoring CH:CHROMATOGRAPHY_SUMMARY (SIM) mode, and untargeted samples in full scan (SCAN) mode with the mass range CH:CHROMATOGRAPHY_SUMMARY of 45-200 amu. The mass spectrometer was operated in electron impact ionization CH:CHROMATOGRAPHY_SUMMARY mode with 70 eV ionization energy, and transfer line, ion source, and quadrupole CH:CHROMATOGRAPHY_SUMMARY temperatures of 250, 280 and 280, respectively. For details on SIM and CH:CHROMATOGRAPHY_SUMMARY significantly altered, identified SCAN compounds, see Table 1. All samples were CH:CHROMATOGRAPHY_SUMMARY analysed within 6 days of collection. The oven program for both SIM and SCAN CH:CHROMATOGRAPHY_SUMMARY analyses were identical and are as follows: 35 ˚C for 2 min, 10 ˚C/min to 155 CH:CHROMATOGRAPHY_SUMMARY ˚C, 1 ˚C/min to 131 ˚C and 25 ˚C/min to 250 with a 5 min 30 second hold. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME HP GCD 1800B CH:COLUMN_NAME Agilent PoraBOND Q (25m x 0.32mm x 0.5um) CH:SOLVENT_A NA CH:SOLVENT_B NA CH:FLOW_GRADIENT NA CH:FLOW_RATE 10ml/min CH:COLUMN_TEMPERATURE 250 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent/HP 5972 MSD MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Calibration was performed using standard gases (BOC Specialty Gases, Woking, MS:MS_COMMENTS UK). Linear regression of calibration curves confirmed strong, positive linear MS:MS_COMMENTS relationships between observed compound peak areas and moles of gas injected for MS:MS_COMMENTS each VOC (r2 > 0.9 in all cases). For compounds not purchased in gaseous state MS:MS_COMMENTS (BOC Specialty gases, as above), 1–2 mL of compound in liquid phase was MS:MS_COMMENTS injected neat into butyl sealed Wheaton-style glass vials (100 mL) and allowed MS:MS_COMMENTS to equilibrate for 1 h. 1 mL of headspace air was then removed from neat vial MS:MS_COMMENTS headspace using a gas tight syringe (Trajan, SGE) and injected into the MS:MS_COMMENTS headspace of a second 100 mL butyl sealed Wheaton-style glass vial. This was MS:MS_COMMENTS then repeated, and 1 mL of the 2nd serial dilution vial was injected into the MS:MS_COMMENTS GCMS system with 29 mL of lab air to give ppb concentrations. This was performed MS:MS_COMMENTS for methanethiol (MeSH (SPEXorganics, St Neots, UK)), isoprene (Alfa Aesar, Ward MS:MS_COMMENTS Hill, MA, USA), acetone (Sigma-Aldrich, Burlington, MA, USA), 2- & 3-methyl MS:MS_COMMENTS pentane and n-hexane (Thermo Scientific, Waltham, MA, USA). Reported compounds MS:MS_COMMENTS detected by the GC/-MS were confirmed by matching retention times and MS:MS_COMMENTS mass–charge (m/z) ratios with known standards. Equation 1: [VOC](ppt)=(CF x MS:MS_COMMENTS 〖10〗^12 x Peak area x Calibration slope)/n Equation 1 outlines the approach MS:MS_COMMENTS to calculating VOC concentrations in parts-per-trillion-by-volume, or pptv. Here MS:MS_COMMENTS Peak area refers to the combined peak areas for the mass-charge ratios MS:MS_COMMENTS identified in Table 1. Multiplying Peak areas by their associated calibration MS:MS_COMMENTS curves (Calibration Slope) generate molar amounts which, when divided by the MS:MS_COMMENTS number of moles of headspace air injected (n), generate a unitless (moles MS:MS_COMMENTS compound/moles of air) ratio. Pptv concentrations are then obtained by MS:MS_COMMENTS multiplying this unitless ratio by 1x1012. For clarity, MS:MS_COMMENTS part-per-billion-by-volume values would be obtained by multiplying the unitless MS:MS_COMMENTS ratios by 1x109, or one billion. Sample VOC concentrations were then normalised MS:MS_COMMENTS to CFC-11 concentrations (240 parts-per-trillion-by-volume (pptv)) through MS:MS_COMMENTS multiplication by a “correction factor”, or CF, Equation 1). CFC-11 was used MS:MS_COMMENTS as an internal standard, since atmospheric concentrations of CFC-11 are globally MS:MS_COMMENTS consistent and stable (Redeker et al., 2007). Quantification of Styrene was done MS:MS_COMMENTS as above but normalisation to CFC-11 was not possible under flushed, hypoxic MS:MS_COMMENTS conditions. NEGATIVE VALUES IN DATA SHOW CONSUMPTION OVER TIME. VARIATION IN MS:MS_COMMENTS SCALE BETWEEN MEDIA SAMPLES ARE DUE TO NORMALISATION OF CELLULAR DATA TO MS:MS_COMMENTS PROTEIN. AS DESCRIBED, MEDIA VALUES ARE SUBTRACTED FROM CELLULAR DATA PRIOR TO MS:MS_COMMENTS NORMALISATION AND EXPRESSED AS PG/HR/UG. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS pg/hr/ug and g/hr for media MS_METABOLITE_DATA_START Samples c1 c2 c3 c4 c5 c6 h1 h2 h3 h4 h5 h6 m1 m2 m3 m4 m5 m6 hm1 hm2 hm3 hm4 hm5 hm6 Factors Sample Type:Cells | Treatment:Control Sample Type:Cells | Treatment:Control Sample Type:Cells | Treatment:Control Sample Type:Cells | Treatment:Control Sample Type:Cells | Treatment:Control Sample Type:Cells | Treatment:Control Sample Type:Cells | Treatment:Hypoxia Sample Type:Cells | Treatment:Hypoxia Sample Type:Cells | Treatment:Hypoxia Sample Type:Cells | Treatment:Hypoxia Sample Type:Cells | Treatment:Hypoxia Sample Type:Cells | Treatment:Hypoxia Sample Type:Media | Treatment:Control Sample Type:Media | Treatment:Control Sample Type:Media | Treatment:Control Sample Type:Media | Treatment:Control Sample Type:Media | Treatment:Control Sample Type:Media | Treatment:Control Sample Type:Media | Treatment:Hypoxia Sample Type:Media | Treatment:Hypoxia Sample Type:Media | Treatment:Hypoxia Sample Type:Media | Treatment:Hypoxia Sample Type:Media | Treatment:Hypoxia Sample Type:Media | Treatment:Hypoxia Methyl Chloride 2.61 1.72 3.1 0.412 0.246 0.159 2.56434 1.37297 3.4969 3.99687 1.46855 6.32169 -5.6E-11 1.95E-11 -3.2E-11 6E-10 1.49E-10 1.19E-09 -1.72E-09 1.49E-10 1.22E-09 -5.98E-10 3.74E-09 -1.59E-10 Methyl Bromide 0.0688 0.0374 0.164 -0.0044 -0.00218 0.0562 -0.32024 -0.21665 -0.4076 -0.68796 -0.25646 -0.35911 -3.1E-12 -5E-11 -1.6E-11 2.73E-11 2E-13 5.77E-11 5.2E-10 2E-13 -4.98E-11 5.19E-12 5.67E-11 -5.47E-11 Methyl Iodide -0.25 -0.435 -0.0682 -0.294 -0.415 -0.898 -0.55367 -0.26582 -0.6111 -1.1995 -0.38518 0.368757 1.11E-09 4.23E-10 1.34E-09 3.59E-10 2.34E-10 3.38E-10 3.18E-10 2.34E-10 7.88E-11 1.92E-10 1.08E-11 -5.23E-12 Dimethyl Sulfide 0.323 0.242 0.625 0.354 0.0873 0.0747 0.20453 0.110079 0.22562 0.335932 0.141956 0.268028 -5.1E-11 1.84E-11 3.16E-11 5.27E-11 -2.9E-11 -3.4E-10 0 -2.9E-11 4.16E-11 -3.88E-11 -9.85E-10 -3.64E-09 Isoprene 0.428 0.32 -0.0481 -0.104 -0.154 0.118 0.481835 0.0738151 0.70204 -0.059799 0.441715 0.0932868 -2.3E-10 2.02E-11 9.96E-10 1.65E-10 -2.3E-09 1.57E-09 -4.71E-11 -2.3E-09 -1.89E-10 -1.55E-12 1.49E-10 2.09E-10 Chloroform -0.104 -0.451 -0.101 -0.62 -0.412 -0.0302 2.30228 1.66244 1.8918 3.25423 1.19027 1.24022 -1.3E-10 4.51E-11 -2.5E-11 -7.4E-11 9.09E-11 -4.9E-10 -1.11E-11 9.09E-11 -6.24E-10 -1.62E-11 1.1E-09 -1.39E-09 Acetone -49.9 116 -35.5 -7.34 -10.4 7.99 19.5694979 -65.161592 8.946913405 50.24965166 3.639117249 -5.562833308 0.000000104 0.000000106 8.23E-08 7.72425E-08 7.22105E-08 1.11894E-07 -9.21E-10 1.22E-09 1.87E-09 -2.08E-10 -9.91E-10 -2.71E-09 2 methyl pentane 1.13 0.0944 0.0303 -0.595 0.123 -0.0467 0.662050292 0.779027602 0.384966679 0.472956307 -0.121430636 -0.801541663 -1.01E-10 8.54E-10 -9E-11 5.66E-10 5.07E-10 5.21E-10 -4.25E-10 2.49E-10 -6.85E-09 -6.56E-10 3.39E-10 -2.31E-10 3 methyl pentane -2 23.3 0.898 -0.138 -1.44 4.16 13.03386692 -5.978676072 0.903350139 -20.0137048 73.69060466 -3.332717179 3.99E-10 3.81E-09 3.38E-09 8.64E-10 -8.24E-11 -1.34E-09 -2.68E-09 1.67E-09 -6.4E-09 1.25E-09 1.98E-09 -3.58E-09 n-hexane 0.269 0.402 1.98 1.22 5.97 16.9 -53.0481823 -39.6221829 -39.4898174 -38.2154298 -62.07532572 -39.84857404 -6.82E-11 1.35E-09 -1.57E-09 2.66E-09 -1.39E-09 4.72E-11 -4.39E-09 1.82E-10 -3.15E-09 -1.46E-09 1.92E-10 1.75E-10 Styrene ND ND ND ND ND ND 3.34E-08 1.52E-08 8.6E-09 2.15E-08 1.23E-08 ND ND ND ND ND ND ND 3.48E-09 -4.03E-09 -2.2E-09 0 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Pubchem ID Methyl Chloride 6327 Methyl Bromide 6323 Methyl Iodide 6328 Dimethyl Sulfide 1068 Isoprene 6557 Chloroform 6212 Acetone 180 2 methyl pentane 7892 3 methyl pentane 7282 n-hexane 8058 Styrene 7501 METABOLITES_END #END