Summary of Study ST004056

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 PR002546. The data can be accessed directly via it's Project DOI: 10.21228/M8JK13 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST004056
Study Title	Analysis of volatile oxidized lipids released during ferroptosis
Study Summary	Ferroptosis, an iron-dependent cell death mechanism characterized by excessive lipid peroxidation, has been implicated in numerous human diseases and organ pathologies. However, current detection methods necessitate invasive tissue sampling to assess lipid peroxidation, making the non-invasive detection of ferroptosis in human subjects extremely challenging. The present study employed oxidative volatolomics to comprehensively characterize volatile oxidized lipids (VOLs) produced during ferroptosis. Ferroptosis was induced in cultured HepG2 cells by RSL3 treatment under two distinct conditions: (screening 1) incubation with ¹⁸O₂/H₂¹⁸O-enriched medium and (screening 2) supplementation with d₅-labeled polyunsaturated fatty acids (d₅-PUFA). The resulting volatile oxidized lipids were analyzed using thermal desorption–gas chromatography–mass spectrometry (TD-GC/MS).
Institute	Kyoto University
Last Name	Matsuoka
First Name	Yuta
Address	Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
Email	matsuoka.yuta.6r@kyoto-u.ac.jp
Phone	+81757534884
Submit Date	2025-07-14
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	GC-MS
Release Date	2025-08-14
Release Version	1

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Project:

Project ID:	PR002546
Project DOI:	doi: 10.21228/M8JK13
Project Title:	Monitoring ferroptosis: Iron-driven volatile oxidized lipids as noninvasive biomarkers
Project Summary:	Ferroptosis, an iron-dependent cell death mechanism characterized by excessive lipid peroxidation, has been implicated in numerous human diseases and organ pathologies. However, current detection methods necessitate invasive tissue sampling to assess lipid peroxidation, making the non-invasive detection of ferroptosis in human subjects extremely challenging. The present study employed oxidative volatolomics to comprehensively characterize volatile oxidized lipids (VOLs) produced during ferroptosis. Polyunsaturated fatty acid (PUFA)-derived VOLs were generated via iron-dependent LPO and were released extracellularly as ferroptosis progressed.
Institute:	Kyoto University
Department:	Graduate School of Medicine
Laboratory:	Center for Cancer Immunotherapy and Immunobiology (CCII)
Last Name:	Matsuoka
First Name:	Yuta
Address:	Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
Email:	matsuoka.yuta.6r@kyoto-u.ac.jp
Phone:	0757534884

Subject:

Subject ID:	SU004202
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

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

mb_sample_id	local_sample_id	Sample source	Treatment
SA469168	sample1	HepG2	Control
SA469169	sample2	HepG2	Control
SA469170	sample3	HepG2	Control
SA469171	sample4	HepG2	RSL3
SA469172	sample5	HepG2	RSL3
SA469173	sample6	HepG2	RSL3
SA469174	sample7	HepG2	RSL3/18O-labeling
SA469175	sample8	HepG2	RSL3/18O-labeling
SA469176	sample9	HepG2	RSL3/18O-labeling
SA469177	sample10	HepG2	RSL3/d5-PUFA-labeling
SA469178	sample11	HepG2	RSL3/d5-PUFA-labeling
SA469179	sample12	HepG2	RSL3/d5-PUFA-labeling

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO004195
Collection Summary:	HepG2 cells (human hepatocellular carcinoma cells) were purchased from the American Type Culture Collection (Manassas, VA, USA). HepG2 cells were cultured in high-glucose Dulbecco’s modified eagle medium (DMEM). All growth media were supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and streptomycin. Cultured HepG2 cells were maintained at 37 °C in a humidified incubator with a 5% CO2 atmosphere, passaged for < 6 months, and were not further tested or authenticated by the authors.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR004211
Treatment Summary:	Screening-1 (18O2/H218O labeling); HepG2 cells (1 × 10⁶ cells) were seeded into culture flasks and incubated for 24 hours. Subsequently, the culture medium was replaced with DMEM prepared using H218O and supplemented with 10% FBS. The flask was then hermetically sealed, and 18O2-enriched air (composition: N2 74%, 18O2 21%, and CO2 5%) generated using the CUBE GM-X3 (FCON CO., LTD., Kochi, Japan) was introduced into the flask at a flow rate of 50 mL/min for 10 minutes via a syringe, thereby replacing the internal atmosphere with ¹⁸O₂ air. Following this, a ferroptosis inducer: RSL3 was added, and after a defined incubation period, headspace gas above the cell culture was collected. Sampling was conducted using the SP209-1000Dual device (GL Sciences Inc., Tokyo, Japan) at a flow rate of 50 mL/min for 30 min, and the volatiles were trapped in inert-coated biomonitoring tubes (Markes International, Llantrisant, UK). The collected samples were subsequently analyzed using thermal desorption–gas chromatography/high-resolution mass spectrometry (TD-GC/HRMS). Screening-2 (d-labeled PUFA labeling); HepG2 cells (5 × 105 cells) were seeded into culture flasks and incubated for 24 hours. Subsequently, d-labeled PUFAs (FA20:4-d5, FA18:2-d4, FA20:4-d8, and FA22:6-d5) were added at a final concentration of 20 µM, followed by an additional 24-hour incubation. After this treatment, the culture medium containing the d-labeled PUFAs was replaced with standard DMEM, and ferroptosis induction, gas collection, and analysis were performed in the same manner as described in Screening-1.

Treatment ID:

TR004211

Treatment Summary:

Screening-1 (18O2/H218O labeling); HepG2 cells (1 × 10⁶ cells) were seeded into culture flasks and incubated for 24 hours. Subsequently, the culture medium was replaced with DMEM prepared using H218O and supplemented with 10% FBS. The flask was then hermetically sealed, and 18O2-enriched air (composition: N2 74%, 18O2 21%, and CO2 5%) generated using the CUBE GM-X3 (FCON CO., LTD., Kochi, Japan) was introduced into the flask at a flow rate of 50 mL/min for 10 minutes via a syringe, thereby replacing the internal atmosphere with ¹⁸O₂ air. Following this, a ferroptosis inducer: RSL3 was added, and after a defined incubation period, headspace gas above the cell culture was collected. Sampling was conducted using the SP209-1000Dual device (GL Sciences Inc., Tokyo, Japan) at a flow rate of 50 mL/min for 30 min, and the volatiles were trapped in inert-coated biomonitoring tubes (Markes International, Llantrisant, UK). The collected samples were subsequently analyzed using thermal desorption–gas chromatography/high-resolution mass spectrometry (TD-GC/HRMS). Screening-2 (d-labeled PUFA labeling); HepG2 cells (5 × 105 cells) were seeded into culture flasks and incubated for 24 hours. Subsequently, d-labeled PUFAs (FA20:4-d5, FA18:2-d4, FA20:4-d8, and FA22:6-d5) were added at a final concentration of 20 µM, followed by an additional 24-hour incubation. After this treatment, the culture medium containing the d-labeled PUFAs was replaced with standard DMEM, and ferroptosis induction, gas collection, and analysis were performed in the same manner as described in Screening-1.

Sample Preparation:

Sampleprep ID:	SP004208
Sampleprep Summary:	The TD tubes were analyzed using a TD-GC-HRMS system. The TD injection system was a TD100-xr (Markes International, California, USA) equipped with a Tenax TA focusing trap. The sample path was set to 230 °C, and the flow rate was adjusted to 10:1 in split mode. The cold focusing trap was set to -30 °C, and the trap desorption was performed at 280 °C.

Combined analysis:

Analysis ID	AN006703
Chromatography ID	CH005093
MS ID	MS006402
Analysis type	MS
Chromatography type	GC
Chromatography system	Thermo Fisher TRACE 1610 Gas Chromatograph
Column	Thermo Fisher TG-5SILMS (60 m x 0.25 mm, 0.25 um )
MS Type	EI
MS instrument type	Orbitrap
MS instrument name	Orbitrap Exploris GC 240
Ion Mode	POSITIVE
Units	Peak area

Chromatography:

Chromatography ID:	CH005093
Chromatography Summary:	GC column: TG-5SILMS (60 m, 0.25 mm ID, 0.25 µm FT, Thermo Fisher Scientific)
Instrument Name:	Thermo Fisher TRACE 1610 Gas Chromatograph
Column Name:	Thermo Fisher TG-5SILMS (60 m x 0.25 mm, 0.25 um )
Column Temperature:	The ramped oven program was set as follows: 30 °C (5 min hold), 5 °C/min to 150 °C, 10 °C/min to 280 °C (20 min hold).
Flow Gradient:	none (GCMS)
Flow Rate:	The carrier flow was set to 1 mL/min with helium.
Solvent A:	none (GCMS)
Solvent B:	none (GCMS)
Chromatography Type:	GC

MS:

MS ID:	MS006402
Analysis ID:	AN006703
Instrument Name:	Orbitrap Exploris GC 240
Instrument Type:	Orbitrap
MS Type:	EI
MS Comments:	An Orbitrap Exploris GC 240 mass spectrometer (Thermo Fisher Scientific) was utilized. Data acquisition was performed using the full scan mode in the m/z 35–450 range and a resolving power of 60,000 at m/z 200. The HRMS data were analyzed using the Compound Discoverer software (Thermo Fisher Scientific).
Ion Mode:	POSITIVE