#METABOLOMICS WORKBENCH yuta624_20250714_231932 DATATRACK_ID:6171 STUDY_ID:ST004056 ANALYSIS_ID:AN006703 PROJECT_ID:PR002546
VERSION             	1
CREATED_ON             	July 21, 2025, 9:33 pm
#PROJECT
PR:PROJECT_TITLE                 	Monitoring ferroptosis: Iron-driven volatile oxidized lipids as noninvasive
PR:PROJECT_TITLE                 	biomarkers
PR:PROJECT_SUMMARY               	Ferroptosis, an iron-dependent cell death mechanism characterized by excessive
PR:PROJECT_SUMMARY               	lipid peroxidation, has been implicated in numerous human diseases and organ
PR:PROJECT_SUMMARY               	pathologies. However, current detection methods necessitate invasive tissue
PR:PROJECT_SUMMARY               	sampling to assess lipid peroxidation, making the non-invasive detection of
PR:PROJECT_SUMMARY               	ferroptosis in human subjects extremely challenging. The present study employed
PR:PROJECT_SUMMARY               	oxidative volatolomics to comprehensively characterize volatile oxidized lipids
PR:PROJECT_SUMMARY               	(VOLs) produced during ferroptosis. Polyunsaturated fatty acid (PUFA)-derived
PR:PROJECT_SUMMARY               	VOLs were generated via iron-dependent LPO and were released extracellularly as
PR:PROJECT_SUMMARY               	ferroptosis progressed.
PR:INSTITUTE                     	Kyoto University
PR:DEPARTMENT                    	Graduate School of Medicine
PR:LABORATORY                    	Center for Cancer Immunotherapy and Immunobiology (CCII)
PR:LAST_NAME                     	Matsuoka
PR:FIRST_NAME                    	Yuta
PR:ADDRESS                       	Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
PR:EMAIL                         	matsuoka.yuta.6r@kyoto-u.ac.jp
PR:PHONE                         	0757534884
#STUDY
ST:STUDY_TITLE                   	Analysis of volatile oxidized lipids released during ferroptosis
ST:STUDY_SUMMARY                 	Ferroptosis, an iron-dependent cell death mechanism characterized by excessive
ST:STUDY_SUMMARY                 	lipid peroxidation, has been implicated in numerous human diseases and organ
ST:STUDY_SUMMARY                 	pathologies. However, current detection methods necessitate invasive tissue
ST:STUDY_SUMMARY                 	sampling to assess lipid peroxidation, making the non-invasive detection of
ST:STUDY_SUMMARY                 	ferroptosis in human subjects extremely challenging. The present study employed
ST:STUDY_SUMMARY                 	oxidative volatolomics to comprehensively characterize volatile oxidized lipids
ST:STUDY_SUMMARY                 	(VOLs) produced during ferroptosis. Ferroptosis was induced in cultured HepG2
ST:STUDY_SUMMARY                 	cells by RSL3 treatment under two distinct conditions: (screening 1) incubation
ST:STUDY_SUMMARY                 	with ¹⁸O₂/H₂¹⁸O-enriched medium and (screening 2) supplementation with
ST:STUDY_SUMMARY                 	d₅-labeled polyunsaturated fatty acids (d₅-PUFA). The resulting volatile
ST:STUDY_SUMMARY                 	oxidized lipids were analyzed using thermal desorption–gas
ST:STUDY_SUMMARY                 	chromatography–mass spectrometry (TD-GC/MS).
ST:INSTITUTE                     	Kyoto University
ST:LAST_NAME                     	Matsuoka
ST:FIRST_NAME                    	Yuta
ST:ADDRESS                       	Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
ST:EMAIL                         	matsuoka.yuta.6r@kyoto-u.ac.jp
ST:PHONE                         	+81757534884
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#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           	-	sample1	Sample source:HepG2 | Treatment:Control	RAW_FILE_NAME(Raw file name)=sample1.mzML
SUBJECT_SAMPLE_FACTORS           	-	sample2	Sample source:HepG2 | Treatment:Control	RAW_FILE_NAME(Raw file name)=sample2.mzML
SUBJECT_SAMPLE_FACTORS           	-	sample3	Sample source:HepG2 | Treatment:Control	RAW_FILE_NAME(Raw file name)=sample3.mzML
SUBJECT_SAMPLE_FACTORS           	-	sample4	Sample source:HepG2 | Treatment:RSL3	RAW_FILE_NAME(Raw file name)=sample4.mzML
SUBJECT_SAMPLE_FACTORS           	-	sample5	Sample source:HepG2 | Treatment:RSL3	RAW_FILE_NAME(Raw file name)=sample5.mzML
SUBJECT_SAMPLE_FACTORS           	-	sample6	Sample source:HepG2 | Treatment:RSL3	RAW_FILE_NAME(Raw file name)=sample6.mzML
SUBJECT_SAMPLE_FACTORS           	-	sample7	Sample source:HepG2 | Treatment:RSL3/18O-labeling	RAW_FILE_NAME(Raw file name)=sample7.mzML
SUBJECT_SAMPLE_FACTORS           	-	sample8	Sample source:HepG2 | Treatment:RSL3/18O-labeling	RAW_FILE_NAME(Raw file name)=sample8.mzML
SUBJECT_SAMPLE_FACTORS           	-	sample9	Sample source:HepG2 | Treatment:RSL3/18O-labeling	RAW_FILE_NAME(Raw file name)=sample9.mzML
SUBJECT_SAMPLE_FACTORS           	-	sample10	Sample source:HepG2 | Treatment:RSL3/d5-PUFA-labeling	RAW_FILE_NAME(Raw file name)=sample10.mzML
SUBJECT_SAMPLE_FACTORS           	-	sample11	Sample source:HepG2 | Treatment:RSL3/d5-PUFA-labeling	RAW_FILE_NAME(Raw file name)=sample11.mzML
SUBJECT_SAMPLE_FACTORS           	-	sample12	Sample source:HepG2 | Treatment:RSL3/d5-PUFA-labeling	RAW_FILE_NAME(Raw file name)=sample12.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	HepG2 cells (human hepatocellular carcinoma cells) were purchased from the
CO:COLLECTION_SUMMARY            	American Type Culture Collection (Manassas, VA, USA). HepG2 cells were cultured
CO:COLLECTION_SUMMARY            	in high-glucose Dulbecco’s modified eagle medium (DMEM). All growth media were
CO:COLLECTION_SUMMARY            	supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and
CO:COLLECTION_SUMMARY            	streptomycin. Cultured HepG2 cells were maintained at 37 °C in a humidified
CO:COLLECTION_SUMMARY            	incubator with a 5% CO2 atmosphere, passaged for < 6 months, and were not
CO:COLLECTION_SUMMARY            	further tested or authenticated by the authors.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Screening-1 (18O2/H218O labeling); HepG2 cells (1 × 10⁶ cells) were seeded
TR:TREATMENT_SUMMARY             	into culture flasks and incubated for 24 hours. Subsequently, the culture medium
TR:TREATMENT_SUMMARY             	was replaced with DMEM prepared using H218O and supplemented with 10% FBS. The
TR:TREATMENT_SUMMARY             	flask was then hermetically sealed, and 18O2-enriched air (composition: N2 74%,
TR:TREATMENT_SUMMARY             	18O2 21%, and CO2 5%) generated using the CUBE GM-X3 (FCON CO., LTD., Kochi,
TR:TREATMENT_SUMMARY             	Japan) was introduced into the flask at a flow rate of 50 mL/min for 10 minutes
TR:TREATMENT_SUMMARY             	via a syringe, thereby replacing the internal atmosphere with ¹⁸O₂ air.
TR:TREATMENT_SUMMARY             	Following this, a ferroptosis inducer: RSL3 was added, and after a defined
TR:TREATMENT_SUMMARY             	incubation period, headspace gas above the cell culture was collected. Sampling
TR:TREATMENT_SUMMARY             	was conducted using the SP209-1000Dual device (GL Sciences Inc., Tokyo, Japan)
TR:TREATMENT_SUMMARY             	at a flow rate of 50 mL/min for 30 min, and the volatiles were trapped in
TR:TREATMENT_SUMMARY             	inert-coated biomonitoring tubes (Markes International, Llantrisant, UK). The
TR:TREATMENT_SUMMARY             	collected samples were subsequently analyzed using thermal desorption–gas
TR:TREATMENT_SUMMARY             	chromatography/high-resolution mass spectrometry (TD-GC/HRMS). Screening-2
TR:TREATMENT_SUMMARY             	(d-labeled PUFA labeling); HepG2 cells (5 × 105 cells) were seeded into culture
TR:TREATMENT_SUMMARY             	flasks and incubated for 24 hours. Subsequently, d-labeled PUFAs (FA20:4-d5,
TR:TREATMENT_SUMMARY             	FA18:2-d4, FA20:4-d8, and FA22:6-d5) were added at a final concentration of 20
TR:TREATMENT_SUMMARY             	µM, followed by an additional 24-hour incubation. After this treatment, the
TR:TREATMENT_SUMMARY             	culture medium containing the d-labeled PUFAs was replaced with standard DMEM,
TR:TREATMENT_SUMMARY             	and ferroptosis induction, gas collection, and analysis were performed in the
TR:TREATMENT_SUMMARY             	same manner as described in Screening-1.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The TD tubes were analyzed using a TD-GC-HRMS system. The TD injection system
SP:SAMPLEPREP_SUMMARY            	was a TD100-xr (Markes International, California, USA) equipped with a Tenax TA
SP:SAMPLEPREP_SUMMARY            	focusing trap. The sample path was set to 230 °C, and the flow rate was
SP:SAMPLEPREP_SUMMARY            	adjusted to 10:1 in split mode. The cold focusing trap was set to -30 °C, and
SP:SAMPLEPREP_SUMMARY            	the trap desorption was performed at 280 °C.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	GC column: TG-5SILMS (60 m, 0.25 mm ID, 0.25 µm FT, Thermo Fisher Scientific)
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Thermo Fisher TRACE 1610 Gas Chromatograph
CH:COLUMN_NAME                   	Thermo Fisher TG-5SILMS (60 m x 0.25 mm, 0.25 um )
CH:SOLVENT_A                     	none (GCMS)
CH:SOLVENT_B                     	none (GCMS)
CH:FLOW_GRADIENT                 	none (GCMS)
CH:FLOW_RATE                     	The carrier flow was set to 1 mL/min with helium.
CH:COLUMN_TEMPERATURE            	The ramped oven program was set as follows: 30 °C (5 min hold), 5 °C/min to
CH:COLUMN_TEMPERATURE            	150 °C, 10 °C/min to 280 °C (20 min hold).
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Orbitrap Exploris GC 240
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	EI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	An Orbitrap Exploris GC 240 mass spectrometer (Thermo Fisher Scientific) was
MS:MS_COMMENTS                   	utilized. Data acquisition was performed using the full scan mode in the m/z
MS:MS_COMMENTS                   	35–450 range and a resolving power of 60,000 at m/z 200. The HRMS data were
MS:MS_COMMENTS                   	analyzed using the Compound Discoverer software (Thermo Fisher Scientific).
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Peak area
MS_METABOLITE_DATA_START
Samples	sample1	sample2	sample3	sample4	sample5	sample6	sample7	sample8	sample9	sample10	sample11	sample12
Factors	Sample source:HepG2 | Treatment:Control	Sample source:HepG2 | Treatment:Control	Sample source:HepG2 | Treatment:Control	Sample source:HepG2 | Treatment:RSL3	Sample source:HepG2 | Treatment:RSL3	Sample source:HepG2 | Treatment:RSL3	Sample source:HepG2 | Treatment:RSL3/18O-labeling	Sample source:HepG2 | Treatment:RSL3/18O-labeling	Sample source:HepG2 | Treatment:RSL3/18O-labeling	Sample source:HepG2 | Treatment:RSL3/d5-PUFA-labeling	Sample source:HepG2 | Treatment:RSL3/d5-PUFA-labeling	Sample source:HepG2 | Treatment:RSL3/d5-PUFA-labeling
2-butanone-18O	0	0	0	0	0	0	171684839	33143648	384143024	0	0	0
2-methylfuran-18O	0	0	0	0	0	0	1234053	767313	1261149	0	0	0
3-methylfuran-18O	0	0	0	0	0	0	362666	239856	316652	0	0	0
3-methylbutanal-18O	0	0	0	0	0	0	7302896	893875	996153	0	0	0
1-butanol-18O	0	0	0	0	0	0	10769452	1379659	1747243	0	0	0
2-pentanone-18O	0	0	0	0	0	0	32986681	14632040	92003867	0	0	0
3-pentanone-18O	0	0	0	0	0	0	4345691	2403182	6420066	0	0	0
2-ethylfuran-18O	0	0	0	0	0	0	635926	453448	885153	0	0	0
2-ethylbutanal-18O	0	0	0	0	0	0	618936	774910	720044	0	0	0
2-propylfuran-18O	0	0	0	0	0	0	105518	107469	154018	0	0	0
2-hexanone-18O	0	0	0	0	0	0	4850538	3553499	12969666	0	0	0
cyclopentaone-18O	0	0	0	0	0	0	673256	1413481	795312	0	0	0
hexanal-18O	0	0	0	0	0	0	4582287	1870247	1217198	0	0	0
5-methyl-2-hexanone-18O	0	0	0	0	0	0	443412	343247	824528	0	0	0
cyclohexanol-18O	0	0	0	0	0	0	171229	188759	212558	0	0	0
heptanal-18O	0	0	0	0	0	0	60268	111276	447946	0	0	0
1-octen-3-ol-18O	0	0	0	0	0	0	1723846	1971688	1507631	0	0	0
3-octanone-18O	0	0	0	0	0	0	355466	274882	378009	0	0	0
2-pentylfuran-18O	0	0	0	0	0	0	65385	102370	62756	0	0	0
octanal-18O	0	0	0	0	0	0	31205	68623	197457	0	0	0
nonanal-18O	0	0	0	0	0	0	98600	141963	862872	0	0	0
1-octen-3-ol-d5	0	0	0	0	0	0	0	0	0	138416	8795	106916
2-pentylfuran-d5	0	0	0	0	0	0	0	0	0	52580	34711	48918
2-butylfuran-d5	0	0	0	0	0	0	0	0	0	3637	1917	2401
1-octen-3-one-d5	0	0	0	0	0	0	0	0	0	2939	350	4102
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Retention time, min	quantified m/z
2-butanone-18O	5.038	45.0227
2-methylfuran-18O	5.137	84.0462
3-methylfuran-18O	5.34	84.0462
3-methylbutanal-18O	6.561	59.0378
1-butanol-18O	6.826	59.0383
2-pentanone-18O	7.512	45.0227
3-pentanone-18O	7.855	59.0383
2-ethylfuran-18O	7.89	83.0378
2-ethylbutanal-18O	11.219	74.0613
2-propylfuran-18O	11.355	83.0378
2-hexanone-18O	11.389	45.0227
cyclopentaone-18O	11.45	86.0618
hexanal-18O	11.82	59.0378
5-methyl-2-hexanone-18O	14.088	45.0227
cyclohexanol-18O	15.282	59.0378
heptanal-18O	15.8	59.0378
1-octen-3-ol-18O	18.66	59.0378
3-octanone-18O	18.863	59.0383
2-pentylfuran-18O	19.05	83.0378
octanal-18O	19.49	59.0378
nonanal-18O	22.89	59.0378
1-octen-3-ol-d5	18.56	104.1124
2-pentylfuran-d5	18.925	143.1358
2-butylfuran-d5	15.25	129.1202
1-octen-3-one-d5	18.45	102.0967
METABOLITES_END
#END