Summary of Study ST002448

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 PR001579. The data can be accessed directly via it's Project DOI: 10.21228/M8KX40 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 ID	ST002448
Study Title	Metabolic analysis of primary HPV18-genome containing human foreskin keratinocytes compared to untransfected donor-matched controls.
Study Summary	Six primary HPV18-genome containing human foreskin keratinocyte cell populations and six donor-matched primary untransfected human foreskin keratinocyte cell populations were grown on lethally-irradiated 3T3-J2 fibroblasts. Before harvesting the keratinocytes, the 3T3-J2 fibroblasts were washed off. Cells and spent media were harvested and frozen at -80°C until processing.
Institute	University of Birmingham, UK
Department	Institute of Cancer and Genomic Sciences
Laboratory	Joanna Parish
Last Name	Parish
First Name	Joanna
Address	IBR Wolfson Drive Medical School, University of Birmingham, Edgbaston
Email	J.L.Parish@bham.ac.uk
Phone	+44 (0)121 415 8151
Submit Date	2023-01-18
Num Groups	2
Total Subjects	12
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-12-31
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001579
Project DOI:	doi: 10.21228/M8KX40
Project Title:	Identifying how human papillomavirus (HPV) 18 establishment alters metabolism in primary human foreskin keratinocytes
Project Summary:	HPV18 is a causative agent of many cancers at a range of anatomical sites. However, the development of cancer often takes many years following initial infection and requires the virus to be able to persist and replicate within the host. Measuring metabolic changes immediately after HPV18 establishment will enable early metabolic changes associated with HPV18 persistence to be determined. Here, we compare the metabolic profiles of HPV18 genome-containing primary human foreskin keratinocytes from six donors, compared to donor-matched untransfected controls. The aim was to identify metabolic and lipid changes associated with HPV establishment in primary human foreskin keratinocytes.
Institute:	University of Birmingham, UK
Department:	Institute of Cancer and Genomic Sciences
Last Name:	Parish
First Name:	Joanna
Address:	IBR Wolfson Drive Medical School
Email:	J.L.Parish@bham.ac.uk
Phone:	+44 (0)121 415 8151

Subject:

Subject ID:	SU002537
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Cell Strain Details:	Primary human foreskin keratinocytes
Cell Primary Immortalized:	Primary
Species Group:	Mammals

Factors:

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

mb_sample_id	local_sample_id	HPV Status
SA244773	CLO_9_CON_cells_LIP_POS	control
SA244774	CHES_4_CON_media_LIP_POS	control
SA244775	GEO_8_CON_media_LIP_POS	control
SA244776	HAR_1_CON_cells_LIP_POS	control
SA244777	CLO_9_CON_media_LIP_NEG	control
SA244778	GEO_8_CON_cells_LIP_POS	control
SA244779	OLI_12_CON_media_LIP_POS	control
SA244780	BER_5_CON_media_LIP_NEG	control
SA244781	OLI_12_CON_media_LIP_NEG	control
SA244782	GEO_8_CON_media_LIP_NEG	control
SA244783	BER_5_CON_media_LIP_POS	control
SA244784	CHES_4_CON_media_LIP_NEG	control
SA244785	CLO_9_CON_media_LIP_POS	control
SA244786	HAR_1_CON_media_LIP_POS	control
SA244787	HAR_1_CON_media_LIP_NEG	control
SA244788	CHES_4_CON_cells_LIP_NEG	control
SA244789	GEO_8_CON_cells_HIL_POS	control
SA244790	CLO_9_CON_cells_HIL_POS	control
SA244791	HAR_1_CON_cells_HIL_POS	control
SA244792	CHES_4_CON_media_HIL_NEG	control
SA244793	BER_5_CON_media_HIL_NEG	control
SA244794	HAR_1_CON_media_HIL_NEG	control
SA244795	OLI_12_CON_media_HIL_NEG	control
SA244796	OLI_12_CON_cells_HIL_POS	control
SA244797	BER_5_CON_cells_HIL_POS	control
SA244798	OLI_12_CON_cells_HIL_NEG	control
SA244799	BER_5_CON_cells_HIL_NEG	control
SA244800	HAR_1_CON_cells_HIL_NEG	control
SA244801	CLO_9_CON_cells_HIL_NEG	control
SA244802	CHES_4_CON_cells_HIL_POS	control
SA244803	GEO_8_CON_cells_HIL_NEG	control
SA244804	CLO_9_CON_media_HIL_NEG	control
SA244805	GEO_8_CON_media_HIL_NEG	control
SA244806	HAR_1_CON_cells_LIP_NEG	control
SA244807	OLI_12_CON_cells_LIP_NEG	control
SA244808	CLO_9_CON_cells_LIP_NEG	control
SA244809	GEO_8_CON_cells_LIP_NEG	control
SA244810	BER_5_CON_cells_LIP_POS	control
SA244811	CHES_4_CON_cells_LIP_POS	control
SA244812	BER_5_CON_cells_LIP_NEG	control
SA244813	CHES_4_CON_cells_HIL_NEG	control
SA244814	BER_5_CON_media_HIL_POS	control
SA244815	CHES_4_CON_media_HIL_POS	control
SA244816	OLI_12_CON_media_HIL_POS	control
SA244817	HAR_1_CON_media_HIL_POS	control
SA244818	GEO_8_CON_media_HIL_POS	control
SA244819	CLO_9_CON_media_HIL_POS	control
SA244820	OLI_12_CON_cells_LIP_POS	control
SA244725	OLI_11_HPV_media_LIP_NEG	HPV
SA244726	HAR_2_HPV_media_LIP_NEG	HPV
SA244727	OLI_11_HPV_media_HIL_POS	HPV
SA244728	BER_6_HPV_media_HIL_POS	HPV
SA244729	HAR_2_HPV_media_HIL_POS	HPV
SA244730	CLO_10_HPV_media_HIL_POS	HPV
SA244731	GEO_7_HPV_media_HIL_NEG	HPV
SA244732	OLI_11_HPV_cells_LIP_POS	HPV
SA244733	HAR_2_HPV_media_HIL_NEG	HPV
SA244734	CLO_10_HPV_media_LIP_NEG	HPV
SA244735	CLO_10_HPV_media_HIL_NEG	HPV
SA244736	GEO_7_HPV_media_HIL_POS	HPV
SA244737	CHES_3_HPV_media_HIL_POS	HPV
SA244738	CHES_3_HPV_media_LIP_NEG	HPV
SA244739	GEO_7_HPV_cells_LIP_NEG	HPV
SA244740	CLO_10_HPV_cells_LIP_NEG	HPV
SA244741	CHES_3_HPV_cells_LIP_POS	HPV
SA244742	BER_6_HPV_cells_LIP_POS	HPV
SA244743	HAR_2_HPV_cells_LIP_POS	HPV
SA244744	CLO_10_HPV_cells_LIP_POS	HPV
SA244745	GEO_7_HPV_cells_LIP_POS	HPV
SA244746	CHES_3_HPV_cells_LIP_NEG	HPV
SA244747	OLI_11_HPV_media_HIL_NEG	HPV
SA244748	BER_6_HPV_cells_LIP_NEG	HPV
SA244749	OLI_11_HPV_cells_LIP_NEG	HPV
SA244750	HAR_2_HPV_cells_LIP_NEG	HPV
SA244751	BER_6_HPV_media_LIP_NEG	HPV
SA244752	GEO_7_HPV_media_LIP_NEG	HPV
SA244753	GEO_7_HPV_cells_HIL_NEG	HPV
SA244754	CLO_10_HPV_cells_HIL_NEG	HPV
SA244755	CHES_3_HPV_cells_HIL_POS	HPV
SA244756	BER_6_HPV_cells_HIL_POS	HPV
SA244757	OLI_11_HPV_media_LIP_POS	HPV
SA244758	CLO_10_HPV_media_LIP_POS	HPV
SA244759	GEO_7_HPV_media_LIP_POS	HPV
SA244760	CHES_3_HPV_cells_HIL_NEG	HPV
SA244761	BER_6_HPV_cells_HIL_NEG	HPV
SA244762	OLI_11_HPV_cells_HIL_NEG	HPV
SA244763	HAR_2_HPV_cells_HIL_NEG	HPV
SA244764	OLI_11_HPV_cells_HIL_POS	HPV
SA244765	HAR_2_HPV_media_LIP_POS	HPV
SA244766	CHES_3_HPV_media_LIP_POS	HPV
SA244767	CLO_10_HPV_cells_HIL_POS	HPV
SA244768	BER_6_HPV_media_LIP_POS	HPV
SA244769	HAR_2_HPV_cells_HIL_POS	HPV
SA244770	GEO_7_HPV_cells_HIL_POS	HPV
SA244771	BER_6_HPV_media_HIL_NEG	HPV
SA244772	CHES_3_HPV_media_HIL_NEG	HPV

Showing results 1 to 96 of 96

Showing results 1 to 96 of 96

Collection:

Collection ID:	CO002530
Collection Summary:	Adherent cells were washed in saline, frozen in the 6-well plate and stored at -80°C. Spent media was taken, frozen and stored at -80°C.
Sample Type:	Keratinocytes

Treatment:

Treatment ID:	TR002549
Treatment Summary:	Normal primary HFKs from neonatal foreskin epithelia were transfected with recircularised HPV18 wild type. All cells were grown in the presence of irradiated 3T3-J2 fibroblasts.

Sample Preparation:

Sampleprep ID:	SP002543
Sampleprep Summary:	Metabolite and lipid extraction from cells and resuspension for UHPLC-MS Metabolites and lipids were extracted from cells in 6-well plates using a biphasic methanol/chloroform/water method. 6-well plates were placed on dry ice and 600 uL ice-cold methanol/water solution (ratio 2/0.8, LC-MS grade, Fisher) was added. Cells were dislodged into the liquid using a cell scraper (Corning) and then all liquid and cells were removed into a clean 1.8 mL glass vial (Wheaton). A further 240 μL methanol/water solution (ratio 2/0.8) was added to the same well, scraped again and the liquid and cells were added to the 1.8 mL glass vial. 600 uL chloroform and 300 uL water (both LC-MS grade, Fisher) were added to the glass vial. Sample was vortexed (30 s), incubated on ice (10 min) and centrifuged (2,500-g, 10 min, 4oC). The sample was set at room temperature for 5 min to allow phase partitioning to complete. All the polar (upper) phase was removed into a 2 mL microfuge tube (Eppendorf) and dried in a SpeedVac concentrator (Savant SPD111V230, Thermo Fisher Scientific). All the non-polar (lower) phase was removed into a 2 mL microfuge tube (Eppendorf) and dried on a nitrogen blow down drier (Techne FSC400D, Thermo Fisher Scientific). To create process blank samples, the entire process was done in the absence of cellular material. Polar samples were reconstituted in 120 uL 3/1 acetonitrile /water (all LC-MS grade, VWR) and non-polar samples reconstituted in 3/1 isopropanol/water (all LC-MS grade, Merck). Each sample was vortexed (15 s), centrifuged (20,000-g, 20 min, 4oC) and 80 uL of the supernatant loaded into a low recovery HPLC vial (Chromatography Direct, UK). Quality control (QC) samples were created by pooling 20 uL from each biological samples and then vortexing (30 s) before distributing across 5 low recovery HPLC vials. Metabolite and lipid extraction from media samples For the extraction of polar metabolites, 150 µL of ice-cold acetonitrile (LC-MS grade, Fisher) was added to 50 µL of media. Each sample was vortexed (15 s), centrifuged (20,000-g, 20 min, 4oC) and 80 uL of the supernatant loaded into a low recovery HPLC vial (Chromatography Direct, UK). For the extraction of non-polar metabolites, 150 µL of ice-cold isopropanol (LC-MS grade, Fisher) was added to 50 µL of media. Each sample was vortexed (15 s), centrifuged (20,000-g, 20 min, 4oC) and 80 uL of the supernatant loaded into a low recovery HPLC vial (Chromatography Direct, UK). QC samples were prepared by pooling 50 uL from each media sample, vortexing (30 s), and then distributing 50 uL aliquots across several 2 mL microfuge tubes (Eppendorf). Process blank samples were prepared by adding 50 uL LC-MS grade water to 2 mL microfuge tubes. QC and process blank samples were extracted as for samples (above) for polar and non-polar metabolites separately.

Sampleprep ID:

SP002543

Sampleprep Summary:

Metabolite and lipid extraction from cells and resuspension for UHPLC-MS Metabolites and lipids were extracted from cells in 6-well plates using a biphasic methanol/chloroform/water method. 6-well plates were placed on dry ice and 600 uL ice-cold methanol/water solution (ratio 2/0.8, LC-MS grade, Fisher) was added. Cells were dislodged into the liquid using a cell scraper (Corning) and then all liquid and cells were removed into a clean 1.8 mL glass vial (Wheaton). A further 240 μL methanol/water solution (ratio 2/0.8) was added to the same well, scraped again and the liquid and cells were added to the 1.8 mL glass vial. 600 uL chloroform and 300 uL water (both LC-MS grade, Fisher) were added to the glass vial. Sample was vortexed (30 s), incubated on ice (10 min) and centrifuged (2,500-g, 10 min, 4oC). The sample was set at room temperature for 5 min to allow phase partitioning to complete. All the polar (upper) phase was removed into a 2 mL microfuge tube (Eppendorf) and dried in a SpeedVac concentrator (Savant SPD111V230, Thermo Fisher Scientific). All the non-polar (lower) phase was removed into a 2 mL microfuge tube (Eppendorf) and dried on a nitrogen blow down drier (Techne FSC400D, Thermo Fisher Scientific). To create process blank samples, the entire process was done in the absence of cellular material. Polar samples were reconstituted in 120 uL 3/1 acetonitrile /water (all LC-MS grade, VWR) and non-polar samples reconstituted in 3/1 isopropanol/water (all LC-MS grade, Merck). Each sample was vortexed (15 s), centrifuged (20,000-g, 20 min, 4oC) and 80 uL of the supernatant loaded into a low recovery HPLC vial (Chromatography Direct, UK). Quality control (QC) samples were created by pooling 20 uL from each biological samples and then vortexing (30 s) before distributing across 5 low recovery HPLC vials. Metabolite and lipid extraction from media samples For the extraction of polar metabolites, 150 µL of ice-cold acetonitrile (LC-MS grade, Fisher) was added to 50 µL of media. Each sample was vortexed (15 s), centrifuged (20,000-g, 20 min, 4oC) and 80 uL of the supernatant loaded into a low recovery HPLC vial (Chromatography Direct, UK). For the extraction of non-polar metabolites, 150 µL of ice-cold isopropanol (LC-MS grade, Fisher) was added to 50 µL of media. Each sample was vortexed (15 s), centrifuged (20,000-g, 20 min, 4oC) and 80 uL of the supernatant loaded into a low recovery HPLC vial (Chromatography Direct, UK). QC samples were prepared by pooling 50 uL from each media sample, vortexing (30 s), and then distributing 50 uL aliquots across several 2 mL microfuge tubes (Eppendorf). Process blank samples were prepared by adding 50 uL LC-MS grade water to 2 mL microfuge tubes. QC and process blank samples were extracted as for samples (above) for polar and non-polar metabolites separately.

Chromatography:

Chromatography ID:	CH002949
Chromatography Summary:	HILIC assays used an Accucore150-Amide-HILIC column (100×2.1mm, 2.6μm, ThermoFisher Scientific). Mobile phase A was 10 mM ammonium formate dissolved in acetonitrile/water/formic acid (95:4.9:0.1 (v/v) and mobile phase B was 10 mM ammonium formate dissolved in acetonitrile/water/formic acid (50/49.9/0.1 (v/v)). The gradient elution applied was t=0.0, 1% B; t=1.0, 1% B; t=3.0, 15% B; t=6.0, 50% B; t=9.0, 95% B; t=10.0, 95% B; t=10.5, 1% B; t=14.0, 1% B. All changes were linear (curve = 5) and the flow rate was 0.50 mL/min. Column temperature was 35 °C and injection volume was 2μL. Data were acquired in positive and negative ionisation mode separately (70 – 1050 m/z) with a mass resolution 70,000 (FWHM, m/z 200). Ion source parameters: Sheath gas = 55 arbitrary units, Aux gas = 35 arbitrary units, Sweep gas = 4 arbitrary units, Spray Voltage = 3.2kV (positive ion) / 2.7kV (negative ion), Capillary temp. = 380°C, Aux gas heater temp. = 440°C. Thermo ExactiveTune (2.8 SP1, build 2806) software controlled the instruments and data acquisition. All data were acquired in profile mode.
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Thermo Accucore HILIC (50 x 2.1mm,2.6um)
Column Temperature:	35 °C
Flow Gradient:	gradient elution applied was t=0.0, 1% B; t=1.0, 1% B; t=3.0, 15% B; t=6.0, 50% B; t=9.0, 95% B; t=10.0, 95% B; t=10.5, 1% B; t=14.0, 1% B. All changes were linear (curve = 5)
Flow Rate:	0.50 mL/min
Solvent A:	95% acetonitrile/5% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:	50% acetonitrile/50% water; 0.1% formic acid; 10 mM ammonium formate
Chromatography Type:	HILIC

Chromatography ID:	CH002950
Chromatography Summary:	Lipid assays used a reversed-phase Hypersil GOLD C18 column (100×2.1 mm, 1.9μm; Thermo Fisher Scientific). Mobile phase A was 10 mM ammonium formate dissolved in acetonitrile/water/formic acid (60:39.9:0.1 (v/v) and mobile phase B was 10 mM ammonium formate dissolved in isopropanol/acetonitrile/water/formic acid (85.5/9.5/4.9/0.1 (v/v)). The gradient elution applied was t=0.0, 20% B; t=0.5, 20% B, t=8.5, 100% B; t=9.5, 100% B; t=11.5, 20% B; t=14.0, 20% B. All changes were linear (curve = 5) and the flow rate was 0.40 mL/min. Column temperature was 55 °C and injection volume was 2μL. Data were acquired in positive and negative ionisation mode separately (150 –2000 m/z) with a mass resolution 70,000 (FWHM, m/z 200). Ion source parameters: Sheath gas = 48 arbitrary units, Aux gas = 15 arbitrary units, Sweep gas = 0 arbitrary units, Spray Voltage = 3.2kV (positive ion) / 2.7kV (negative ion), Capillary temp. = 380°C, Aux gas heater temp. = 450°C. Thermo ExactiveTune (2.8 SP1, build 2806) software controlled the instruments and data acquisition. All data were acquired in profile mode.
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um)
Column Temperature:	55 °C
Flow Gradient:	gradient elution applied was t=0.0, 20% B; t=0.5, 20% B, t=8.5, 100% B; t=9.5, 100% B; t=11.5, 20% B; t=14.0, 20% B. All changes were linear (curve = 5)
Flow Rate:	0.40 mL/min
Solvent A:	60% acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:	85.5% isopropanol/9.5% acetonitrile/5% water; 0.1% formic acid; 10 mM ammonium formate
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN003989
Analysis Type:	MS
Chromatography ID:	CH002949
Has Mz:	1
Has Rt:	1
Rt Units:	Seconds
Results File:	ST002448_AN003989_Results.txt
Units:	Peak area

Analysis ID:	AN003990
Analysis Type:	MS
Chromatography ID:	CH002949
Has Mz:	1
Has Rt:	1
Rt Units:	Seconds
Results File:	ST002448_AN003990_Results.txt
Units:	Peak area

Analysis ID:	AN003991
Analysis Type:	MS
Chromatography ID:	CH002949
Has Mz:	1
Has Rt:	1
Rt Units:	Seconds
Results File:	ST002448_AN003991_Results.txt
Units:	Peak area

Analysis ID:	AN003992
Analysis Type:	MS
Chromatography ID:	CH002949
Has Mz:	1
Has Rt:	1
Rt Units:	Seconds
Results File:	ST002448_AN003992_Results.txt
Units:	Peak area

Analysis ID:	AN003993
Analysis Type:	MS
Chromatography ID:	CH002950
Has Mz:	1
Has Rt:	1
Rt Units:	Seconds
Results File:	ST002448_AN003993_Results.txt
Units:	Peak area

Analysis ID:	AN003994
Analysis Type:	MS
Chromatography ID:	CH002950
Has Mz:	1
Has Rt:	1
Rt Units:	Seconds
Results File:	ST002448_AN003994_Results.txt
Units:	Peak area

Analysis ID:	AN003995
Analysis Type:	MS
Chromatography ID:	CH002950
Has Mz:	1
Has Rt:	1
Rt Units:	Seconds
Results File:	ST002448_AN003995_Results.txt
Units:	Peak area

Analysis ID:	AN003996
Analysis Type:	MS
Chromatography ID:	CH002950
Has Mz:	1
Has Rt:	1
Rt Units:	Seconds
Results File:	ST002448_AN003996_Results.txt
Units:	Peak area