#METABOLOMICS WORKBENCH Dmitri_20230825_211119 DATATRACK_ID:4249 STUDY_ID:ST002839 ANALYSIS_ID:AN004645 PROJECT_ID:PR001778
VERSION             	1
CREATED_ON             	August 30, 2023, 4:35 am
#PROJECT
PR:PROJECT_TITLE                 	Metabolites alteration during ferroptotic process in tumor cells
PR:PROJECT_SUMMARY               	Targeting ferroptosis, an iron-dependent form of regulated cell death triggered
PR:PROJECT_SUMMARY               	by the lethal overload of lipid peroxides, in cancer therapy is impeded by our
PR:PROJECT_SUMMARY               	limited understanding of the intersection of tumour’s metabolic feature and
PR:PROJECT_SUMMARY               	ferroptosis vulnerability. In the presented study, we performed metabolomics for
PR:PROJECT_SUMMARY               	cancer cell lines treated by ferroptosis inducer RSL3 or control DMSO.
PR:INSTITUTE                     	Zhongshan Hospital Fudan University
PR:LAST_NAME                     	Bi
PR:FIRST_NAME                    	Guoshu
PR:ADDRESS                       	Zhongshan Hospital, Fudan University, No. 180, Fenglin Road, Xuhui District,
PR:ADDRESS                       	Shanghai, China
PR:EMAIL                         	18211210035@fudan.edu.cn
PR:PHONE                         	86 64041990
#STUDY
ST:STUDY_TITLE                   	Metabolic alteration during ferroptotic process in cancer cells.
ST:STUDY_SUMMARY                 	Targeting ferroptosis, an iron-dependent form of regulated cell death triggered
ST:STUDY_SUMMARY                 	by the lethal overload of lipid peroxides, in cancer therapy is impeded by our
ST:STUDY_SUMMARY                 	limited understanding of the intersection of tumour’s metabolic feature and
ST:STUDY_SUMMARY                 	ferroptosis vulnerability. In this study, we performed metabolomics in cancer
ST:STUDY_SUMMARY                 	cell lines pretreated with ferroptosis inducer RSL3 and control DMSO. We noted
ST:STUDY_SUMMARY                 	that the levels of a series of metabolites were significantly impacted by the
ST:STUDY_SUMMARY                 	RSL3 treatment, such as upregulated polyamines including putrescine, spermidine,
ST:STUDY_SUMMARY                 	and spermine. According to our previous study, we proved the pro-ferroptotic
ST:STUDY_SUMMARY                 	feature of polyamines in tumor cells, which was derived from the H2O2 produced
ST:STUDY_SUMMARY                 	during the polyamine metabolic process. This finding is consistent with our
ST:STUDY_SUMMARY                 	RNA-Seq data indicating upregulated ODC1 in the ferroptotic process. Therefore,
ST:STUDY_SUMMARY                 	it could be speculated that the RSL3-induced polyamine production leads to a
ST:STUDY_SUMMARY                 	positive-feedback loop that amplifies ferroptosis in tumor cells.
ST:INSTITUTE                     	Zhongshan Hospital Fudan University
ST:LAST_NAME                     	Bi
ST:FIRST_NAME                    	Guoshu
ST:ADDRESS                       	Zhongshan Hospital, Fudan University, No. 180, Fenglin Road, Xuhui District,
ST:ADDRESS                       	Shanghai, China
ST:EMAIL                         	18211210035@fudan.edu.cn
ST:PHONE                         	86 64041990
#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           	-	ND1	Treatment:Control	genotype=Wild-type; RAW_FILE_NAME=ND1.raw
SUBJECT_SAMPLE_FACTORS           	-	ND2	Treatment:Control	genotype=Wild-type; RAW_FILE_NAME=ND2.raw
SUBJECT_SAMPLE_FACTORS           	-	NR1	Treatment:RSL3	genotype=Wild-type; RAW_FILE_NAME=NR1.raw
SUBJECT_SAMPLE_FACTORS           	-	NR2	Treatment:RSL3	genotype=Wild-type; RAW_FILE_NAME=NR2.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	For cell samples, after being washed with ice-cold PBS, the cells were collected
CO:COLLECTION_SUMMARY            	using a cell scraper and centrifuged for 5 min at 1,000 g. The samples were
CO:COLLECTION_SUMMARY            	homogenized in liquid N2 for 1 min and defrosted in 4°C.
CO:SAMPLE_TYPE                   	Tumor cells
#TREATMENT
TR:TREATMENT_SUMMARY             	A549 cells cultured in DMEM medium were treated with 0.5 uM RSL3 or DMSO for 24
TR:TREATMENT_SUMMARY             	h.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	After homogenization in liquid N2, 200 μL 80% methyl alcohol was added,
SP:SAMPLEPREP_SUMMARY            	followed by vortex for 1 min, sonication for 30 min at 4°C, and stand for 1 h
SP:SAMPLEPREP_SUMMARY            	at -20°C. Then, the samples were centrifuged at 12,000 rpm for 15 min at 4°C,
SP:SAMPLEPREP_SUMMARY            	and the supernatant was collected for subsequent LC-MS analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	LC
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um)
CH:SOLVENT_A                     	Water containing 0.1% methanoic acid
CH:SOLVENT_B                     	Acetonitrile containing 0.1% methanoic acid
CH:FLOW_GRADIENT                 	Time:(0.01-2-4-8-10-14-15-15.1-16); Solvent A%:
CH:FLOW_GRADIENT                 	(95-95-70-50-20-0-0-95-95);Solvent B%: (5-5-30-50-80-100-100-5-5)
CH:FLOW_RATE                     	0.35 mL/min
CH:COLUMN_TEMPERATURE            	45
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Mass Scan Range 100-1200 Resolution (full scan) 70000 Resolution (HCD MS/MS
MS:MS_COMMENTS                   	scans) 17500 Spray Voltage (V) 3800 Sheath Gas Flow Rate (Arb) 35 Aux Gas Flow
MS:MS_COMMENTS                   	Rate (Arb) 8 Capillary Temperature (°C) 320
MS:MS_RESULTS_FILE               	ST002839_AN004645_Results.txt	UNITS:pmol/l	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END