#METABOLOMICS WORKBENCH dmukha_20240309_152107 DATATRACK_ID:4696 STUDY_ID:ST003151 ANALYSIS_ID:AN005170 PROJECT_ID:PR001941
VERSION             	1
CREATED_ON             	March 27, 2024, 10:47 am
#PROJECT
PR:PROJECT_TITLE                 	The shutdown of NADH oxidation via Respiratory Complex I mimics fatty acid
PR:PROJECT_TITLE                 	biosynthesis inhibition
PR:PROJECT_TYPE                  	LC-MS Quantitative Analysis
PR:PROJECT_SUMMARY               	Proliferating cancer cells actively utilize anabolic processes for biomass
PR:PROJECT_SUMMARY               	production, including de novo biosynthesis of amino acids, nucleotides, and
PR:PROJECT_SUMMARY               	fatty acids. The key enzyme of the fatty acid biosynthesis pathway, fatty acid
PR:PROJECT_SUMMARY               	synthase (FASN), is widely recognized as a promising therapeutic target in
PR:PROJECT_SUMMARY               	cancer and other health conditions. Here, we establish a metabolic signature of
PR:PROJECT_SUMMARY               	FASN inhibition using a panel of pharmacological inhibitors (GSK2194069,
PR:PROJECT_SUMMARY               	TVB-2640, TVB-3166, C75, cerulenin, and Fasnall). We find that the activity of
PR:PROJECT_SUMMARY               	some commonly used FASN inhibitors is inconsistent with the metabolic signature
PR:PROJECT_SUMMARY               	of FASN inhibition (accumulation of malonate, succinate, malonyl coenzyme A,
PR:PROJECT_SUMMARY               	succinyl coenzyme A, and other metabolic perturbations). Moreover, we show that
PR:PROJECT_SUMMARY               	one of these putative FASN inhibitors, Fasnall, is a respiratory Complex I
PR:PROJECT_SUMMARY               	inhibitor that mimics FASN inhibition through NADH accumulation and consequent
PR:PROJECT_SUMMARY               	depletion of the tricarboxylic acid cycle metabolites. We demonstrate that
PR:PROJECT_SUMMARY               	Fasnall impairs tumor growth in several oxidative phosphorylation-dependent
PR:PROJECT_SUMMARY               	cancer models, including combination therapy-resistant melanoma patient-derived
PR:PROJECT_SUMMARY               	xenografts. Fasnall administration does not reproduce neurological side effects
PR:PROJECT_SUMMARY               	in mice reported for other Complex I inhibitors. Our results have significant
PR:PROJECT_SUMMARY               	implications for understanding the FASN role in human health and disease and
PR:PROJECT_SUMMARY               	provide evidence of therapeutic potential for Complex I inhibitors with fast
PR:PROJECT_SUMMARY               	systemic clearance. Our findings also highlight the continuing need for
PR:PROJECT_SUMMARY               	validation of small molecule inhibitors to distinguish high-quality chemical
PR:PROJECT_SUMMARY               	probes and to expand the understanding of their application.
PR:INSTITUTE                     	Wistar Institute
PR:DEPARTMENT                    	Molecular and Cellular Oncogenesis Program, Ellen and Ronald Caplan Cancer
PR:DEPARTMENT                    	Center
PR:LABORATORY                    	Schug's Lab
PR:LAST_NAME                     	Mukha
PR:FIRST_NAME                    	Dzmitry
PR:ADDRESS                       	3601 Spruce St., Philadelphia, Pennsylvania 19104, USA
PR:EMAIL                         	dmukha@wistar.org
PR:PHONE                         	+12154956903
PR:FUNDING_SOURCE                	This work was supported by grants from the National Institutes of Health (NIH)
PR:FUNDING_SOURCE                	National Cancer Institute (NCI) DP2 CA249950-01 (Z.T.S.), NIH NCI P01 CA114046
PR:FUNDING_SOURCE                	(Z.T.S.), Melanoma Research Foundation 717173 (Z.T.S.), and Susan G. Komen
PR:FUNDING_SOURCE                	CCR19608782 (Z.T.S.).
PR:PUBLICATIONS                  	Submission Pending
PR:CONTRIBUTORS                  	Dzmitry Mukha, Jena Dessain, Seamus O’Connor, Katherine Pniewski, Fabrizio
PR:CONTRIBUTORS                  	Bertolazzi, Jeet Patel, Mary Mullins, Zachary T. Schug
#STUDY
ST:STUDY_TITLE                   	BT474 breast cancer cell line grown in 20% D2O-containing RPMI-1640 medium
ST:STUDY_TITLE                   	treated with Fasnall and GSK2194069
ST:STUDY_TYPE                    	Free fatty acid analysis, D2O tracing
ST:STUDY_SUMMARY                 	BT474 breast cancer cell line grown in 20% D2O-containing medium treated with
ST:STUDY_SUMMARY                 	Fasnall and GSK2194069. Cells were grown for 24 h in RPMI-1640 with 10% dialyzed
ST:STUDY_SUMMARY                 	FBS.
ST:INSTITUTE                     	Wistar Institute
ST:DEPARTMENT                    	Molecular and Cellular Oncogenesis Program, Ellen and Ronald Caplan Cancer
ST:DEPARTMENT                    	Center
ST:LABORATORY                    	Schug's Lab
ST:LAST_NAME                     	Mukha
ST:FIRST_NAME                    	Dzmitry
ST:ADDRESS                       	3601 Spruce St, Philadelphia, PA 19104, USA
ST:EMAIL                         	dmukha@wistar.org
ST:PHONE                         	2154956903
ST:NUM_GROUPS                    	7
ST:TOTAL_SUBJECTS                	21
ST:PUBLICATIONS                  	Submission Pending
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:AGE_OR_AGE_RANGE              	60
SU:GENDER                        	Female
SU:CELL_STRAIN_DETAILS           	BT-474, breast cancer cell line
#FACTORS
#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           	-	01_FFA_Blank	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA	RAW_FILE_NAME(Raw File Name)=01_FFA_Blank.raw; RAW_FILE_NAME(Raw File Name)=NEG_01_FFA_Blank.mzXML; RAW_FILE_NAME(Raw File Name )=POS_01_FFA_Blank.mzXML
SUBJECT_SAMPLE_FACTORS           	-	02_FFA_Blank	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA	RAW_FILE_NAME(Raw File Name)=02_FFA_Blank.raw; RAW_FILE_NAME(Raw File Name)=NEG_02_FFA_Blank.mzXML; RAW_FILE_NAME(Raw File Name )=POS_02_FFA_Blank.mzXML
SUBJECT_SAMPLE_FACTORS           	-	03_FFA_Blank	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA	RAW_FILE_NAME(Raw File Name)=03_FFA_Blank.raw; RAW_FILE_NAME(Raw File Name)=NEG_03_FFA_Blank.mzXML; RAW_FILE_NAME(Raw File Name )=POS_03_FFA_Blank.mzXML
SUBJECT_SAMPLE_FACTORS           	-	04_FFA_D2O_BT474_Control	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:Vehicle	RAW_FILE_NAME(Raw File Name)=04_FFA_D2O_BT474_Control.raw; RAW_FILE_NAME(Raw File Name)=NEG_04_FFA_D2O_BT474_Control.mzXML; RAW_FILE_NAME(Raw File Name )=POS_04_FFA_D2O_BT474_Control.mzXML
SUBJECT_SAMPLE_FACTORS           	-	05_FFA_D2O_BT474_GSK0001	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 nM GSK2194069	RAW_FILE_NAME(Raw File Name)=05_FFA_D2O_BT474_GSK0001.raw; RAW_FILE_NAME(Raw File Name)=NEG_05_FFA_D2O_BT474_GSK0001.mzXML; RAW_FILE_NAME(Raw File Name )=POS_05_FFA_D2O_BT474_GSK0001.mzXML
SUBJECT_SAMPLE_FACTORS           	-	06_FFA_D2O_BT474_GSK0040	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 nM GSK2194069	RAW_FILE_NAME(Raw File Name)=06_FFA_D2O_BT474_GSK0040.raw; RAW_FILE_NAME(Raw File Name)=NEG_06_FFA_D2O_BT474_GSK0040.mzXML; RAW_FILE_NAME(Raw File Name )=POS_06_FFA_D2O_BT474_GSK0040.mzXML
SUBJECT_SAMPLE_FACTORS           	-	07_FFA_D2O_BT474_GSK1000	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM GSK2194069	RAW_FILE_NAME(Raw File Name)=07_FFA_D2O_BT474_GSK1000.raw; RAW_FILE_NAME(Raw File Name)=NEG_07_FFA_D2O_BT474_GSK1000.mzXML; RAW_FILE_NAME(Raw File Name )=POS_07_FFA_D2O_BT474_GSK1000.mzXML
SUBJECT_SAMPLE_FACTORS           	-	08_FFA_D2O_BT474_Fasnall01	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM Fasnall	RAW_FILE_NAME(Raw File Name)=08_FFA_D2O_BT474_Fasnall01.raw; RAW_FILE_NAME(Raw File Name)=NEG_08_FFA_D2O_BT474_Fasnall01.mzXML; RAW_FILE_NAME(Raw File Name )=POS_08_FFA_D2O_BT474_Fasnall01.mzXML
SUBJECT_SAMPLE_FACTORS           	-	09_FFA_D2O_BT474_Fasnall05	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:5 uM Fasnall	RAW_FILE_NAME(Raw File Name)=09_FFA_D2O_BT474_Fasnall05.raw; RAW_FILE_NAME(Raw File Name)=NEG_09_FFA_D2O_BT474_Fasnall05.mzXML; RAW_FILE_NAME(Raw File Name )=POS_09_FFA_D2O_BT474_Fasnall05.mzXML
SUBJECT_SAMPLE_FACTORS           	-	10_FFA_D2O_BT474_Fasnall40	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 uM Fasnall	RAW_FILE_NAME(Raw File Name)=10_FFA_D2O_BT474_Fasnall40.raw; RAW_FILE_NAME(Raw File Name)=NEG_10_FFA_D2O_BT474_Fasnall40.mzXML; RAW_FILE_NAME(Raw File Name )=POS_10_FFA_D2O_BT474_Fasnall40.mzXML
SUBJECT_SAMPLE_FACTORS           	-	11_FFA_D2O_BT474_Control	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:Vehicle	RAW_FILE_NAME(Raw File Name)=11_FFA_D2O_BT474_Control.raw; RAW_FILE_NAME(Raw File Name)=NEG_11_FFA_D2O_BT474_Control.mzXML; RAW_FILE_NAME(Raw File Name )=POS_11_FFA_D2O_BT474_Control.mzXML
SUBJECT_SAMPLE_FACTORS           	-	12_FFA_D2O_BT474_GSK0001	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 nM GSK2194069	RAW_FILE_NAME(Raw File Name)=12_FFA_D2O_BT474_GSK0001.raw; RAW_FILE_NAME(Raw File Name)=NEG_12_FFA_D2O_BT474_GSK0001.mzXML; RAW_FILE_NAME(Raw File Name )=POS_12_FFA_D2O_BT474_GSK0001.mzXML
SUBJECT_SAMPLE_FACTORS           	-	13_FFA_D2O_BT474_GSK0040	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 nM GSK2194069	RAW_FILE_NAME(Raw File Name)=13_FFA_D2O_BT474_GSK0040.raw; RAW_FILE_NAME(Raw File Name)=NEG_13_FFA_D2O_BT474_GSK0040.mzXML; RAW_FILE_NAME(Raw File Name )=POS_13_FFA_D2O_BT474_GSK0040.mzXML
SUBJECT_SAMPLE_FACTORS           	-	14_FFA_D2O_BT474_GSK1000	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM GSK2194069	RAW_FILE_NAME(Raw File Name)=14_FFA_D2O_BT474_GSK1000.raw; RAW_FILE_NAME(Raw File Name)=NEG_14_FFA_D2O_BT474_GSK1000.mzXML; RAW_FILE_NAME(Raw File Name )=POS_14_FFA_D2O_BT474_GSK1000.mzXML
SUBJECT_SAMPLE_FACTORS           	-	15_FFA_D2O_BT474_Fasnall01	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM Fasnall	RAW_FILE_NAME(Raw File Name)=15_FFA_D2O_BT474_Fasnall01.raw; RAW_FILE_NAME(Raw File Name)=NEG_15_FFA_D2O_BT474_Fasnall01.mzXML; RAW_FILE_NAME(Raw File Name )=POS_15_FFA_D2O_BT474_Fasnall01.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16_FFA_D2O_BT474_Fasnall05	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:5 uM Fasnall	RAW_FILE_NAME(Raw File Name)=16_FFA_D2O_BT474_Fasnall05.raw; RAW_FILE_NAME(Raw File Name)=NEG_16_FFA_D2O_BT474_Fasnall05.mzXML; RAW_FILE_NAME(Raw File Name )=POS_16_FFA_D2O_BT474_Fasnall05.mzXML
SUBJECT_SAMPLE_FACTORS           	-	17_FFA_D2O_BT474_Fasnall40	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 uM Fasnall	RAW_FILE_NAME(Raw File Name)=17_FFA_D2O_BT474_Fasnall40.raw; RAW_FILE_NAME(Raw File Name)=NEG_17_FFA_D2O_BT474_Fasnall40.mzXML; RAW_FILE_NAME(Raw File Name )=POS_17_FFA_D2O_BT474_Fasnall40.mzXML
SUBJECT_SAMPLE_FACTORS           	-	18_FFA_D2O_BT474_Control	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:Vehicle	RAW_FILE_NAME(Raw File Name)=18_FFA_D2O_BT474_Control.raw; RAW_FILE_NAME(Raw File Name)=NEG_18_FFA_D2O_BT474_Control.mzXML; RAW_FILE_NAME(Raw File Name )=POS_18_FFA_D2O_BT474_Control.mzXML
SUBJECT_SAMPLE_FACTORS           	-	19_FFA_D2O_BT474_GSK0001	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 nM GSK2194069	RAW_FILE_NAME(Raw File Name)=19_FFA_D2O_BT474_GSK0001.raw; RAW_FILE_NAME(Raw File Name)=NEG_19_FFA_D2O_BT474_GSK0001.mzXML; RAW_FILE_NAME(Raw File Name )=POS_19_FFA_D2O_BT474_GSK0001.mzXML
SUBJECT_SAMPLE_FACTORS           	-	20_FFA_D2O_BT474_GSK0040	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 nM GSK2194069	RAW_FILE_NAME(Raw File Name)=20_FFA_D2O_BT474_GSK0040.raw; RAW_FILE_NAME(Raw File Name)=NEG_20_FFA_D2O_BT474_GSK0040.mzXML; RAW_FILE_NAME(Raw File Name )=POS_20_FFA_D2O_BT474_GSK0040.mzXML
SUBJECT_SAMPLE_FACTORS           	-	21_FFA_D2O_BT474_GSK1000	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM GSK2194069	RAW_FILE_NAME(Raw File Name)=21_FFA_D2O_BT474_GSK1000.raw; RAW_FILE_NAME(Raw File Name)=NEG_21_FFA_D2O_BT474_GSK1000.mzXML; RAW_FILE_NAME(Raw File Name )=POS_21_FFA_D2O_BT474_GSK1000.mzXML
SUBJECT_SAMPLE_FACTORS           	-	22_FFA_D2O_BT474_Fasnall01	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM Fasnall	RAW_FILE_NAME(Raw File Name)=22_FFA_D2O_BT474_Fasnall01.raw; RAW_FILE_NAME(Raw File Name)=NEG_22_FFA_D2O_BT474_Fasnall01.mzXML; RAW_FILE_NAME(Raw File Name )=POS_22_FFA_D2O_BT474_Fasnall01.mzXML
SUBJECT_SAMPLE_FACTORS           	-	23_FFA_D2O_BT474_Fasnall05	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:5 uM Fasnall	RAW_FILE_NAME(Raw File Name)=23_FFA_D2O_BT474_Fasnall05.raw; RAW_FILE_NAME(Raw File Name)=NEG_23_FFA_D2O_BT474_Fasnall05.mzXML; RAW_FILE_NAME(Raw File Name )=POS_23_FFA_D2O_BT474_Fasnall05.mzXML
SUBJECT_SAMPLE_FACTORS           	-	24_FFA_D2O_BT474_Fasnall40	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 uM Fasnall	RAW_FILE_NAME(Raw File Name)=24_FFA_D2O_BT474_Fasnall40.raw; RAW_FILE_NAME(Raw File Name)=NEG_24_FFA_D2O_BT474_Fasnall40.mzXML; RAW_FILE_NAME(Raw File Name )=POS_24_FFA_D2O_BT474_Fasnall40.mzXML
SUBJECT_SAMPLE_FACTORS           	-	25_FFA_Blank	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA	RAW_FILE_NAME(Raw File Name)=25_FFA_Blank.raw; RAW_FILE_NAME(Raw File Name)=NEG_25_FFA_Blank.mzXML; RAW_FILE_NAME(Raw File Name )=POS_25_FFA_Blank.mzXML
SUBJECT_SAMPLE_FACTORS           	-	26_FFA_Blank	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA	RAW_FILE_NAME(Raw File Name)=26_FFA_Blank.raw; RAW_FILE_NAME(Raw File Name)=NEG_26_FFA_Blank.mzXML; RAW_FILE_NAME(Raw File Name )=POS_26_FFA_Blank.mzXML
SUBJECT_SAMPLE_FACTORS           	-	27_FFA_Blank	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA	RAW_FILE_NAME(Raw File Name)=27_FFA_Blank.raw; RAW_FILE_NAME(Raw File Name)=NEG_27_FFA_Blank.mzXML; RAW_FILE_NAME(Raw File Name )=POS_27_FFA_Blank.mzXML
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells were seeded in 10 cm Petri dishes. At collection, cells were washed with
CO:COLLECTION_SUMMARY            	PBS three times. Then, 1 ml of methanol was added and cells were scraped from
CO:COLLECTION_SUMMARY            	the surface. All content of the plate was transferred into 13 x 100 mm Pyrex
CO:COLLECTION_SUMMARY            	glass tubes. Lipids were extracted by the Folch method. Dried lipids were
CO:COLLECTION_SUMMARY            	redissolved in 1 ml of 0.3 M KOH solution in 90% methanol and incubated at 85
CO:COLLECTION_SUMMARY            	°C for 1 h. Then, 100 µl of formic acid were added, followed by 800 µl of
CO:COLLECTION_SUMMARY            	hexane for extraction. The hexane phase was transferred to glass LC-MS vials and
CO:COLLECTION_SUMMARY            	dried under the stream of nitrogen. Samples were redissolved in 1 ml of 1:1
CO:COLLECTION_SUMMARY            	methanol:isopropanol.
CO:COLLECTION_PROTOCOL_FILENAME  	DM_free_fatty_acid_analysis_samples.txt
CO:SAMPLE_TYPE                   	Cultured cells
CO:COLLECTION_METHOD             	100% methanol extraction
CO:VOLUMEORAMOUNT_COLLECTED      	1 ml
CO:STORAGE_CONDITIONS            	-80℃
CO:COLLECTION_VIALS              	1.5 ml plastic centrifuge tubes
CO:STORAGE_VIALS                 	1.5 ml plastic centrifuge tubes
#TREATMENT
TR:TREATMENT_SUMMARY             	Cells were grown in 20% D2O-containing RPMI-1640 medium and treated with Fasnall
TR:TREATMENT_SUMMARY             	or GSK2194069 at various concentrations.
TR:TREATMENT_COMPOUND            	Fasnall, GSK2194069
TR:TREATMENT_DOSE                	1 nM - 40 uM
TR:TREATMENT_DOSEDURATION        	24 h
TR:TREATMENT_VEHICLE             	DMSO
TR:CELL_GROWTH_CONTAINER         	6-cm Petri dishes
TR:CELL_MEDIA                    	RPMI-1640
TR:CELL_ENVIR_COND               	37C, 5% CO2
TR:CELL_PCT_CONFLUENCE           	~70%
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Sample preparation for LC-MS free fatty acid analysis Cells were seeded in 10 cm
SP:SAMPLEPREP_SUMMARY            	Petri dishes. At collection, cells were washed with PBS three times. Then, 1 ml
SP:SAMPLEPREP_SUMMARY            	of methanol was added and cells were scraped from the surface. All content of
SP:SAMPLEPREP_SUMMARY            	the plate was transferred into 13 x 100 mm Pyrex glass tubes. Lipids were
SP:SAMPLEPREP_SUMMARY            	extracted by the Folch method. Dried lipids were redissolved in 1 ml of 0.3 M
SP:SAMPLEPREP_SUMMARY            	KOH solution in 90% methanol and incubated at 85 °C for 1 h. Then, 100 µl of
SP:SAMPLEPREP_SUMMARY            	formic acid were added, followed by 800 µl of hexane for extraction. The hexane
SP:SAMPLEPREP_SUMMARY            	phase was transferred to glass LC-MS vials and dried under the stream of
SP:SAMPLEPREP_SUMMARY            	nitrogen. Samples were redissolved in 1 ml of 1:1 methanol:isopropanol.
SP:SAMPLEPREP_PROTOCOL_FILENAME  	DM_free_fatty_acid_analysis_samples.txt
SP:PROCESSING_METHOD             	Lipid saponification in 0.3 M KOH
SP:EXTRACTION_METHOD             	100% methanol
SP:EXTRACT_ENRICHMENT            	None
SP:EXTRACT_CLEANUP               	None
SP:EXTRACT_STORAGE               	-80℃
SP:SAMPLE_RESUSPENSION           	None
SP:SAMPLE_DERIVATIZATION         	None
SP:SAMPLE_SPIKING                	None
SP:SUBCELLULAR_LOCATION          	Cellular lipids
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Dionex Ultimate 3000 RS
CH:COLUMN_NAME                   	Phenomenex Kinetex XB-C18 (150 x 3 mm, 2.6 um)
CH:SOLVENT_A                     	60:40 Acetonitrile/water; 10 mM ammonium formate
CH:SOLVENT_B                     	90:8:2 Isopropanol/acetonitrile/water; 10 mM ammonium formate
CH:FLOW_GRADIENT                 	0-4.5 min, 15-60% B; 4.5-12 min, 60-82% B; 12-12.75 min, 82-95% B; 12.75-16.5
CH:FLOW_GRADIENT                 	min, 95-100% B; 16.5-22.5 min, 100% B; 22.5-22.6 min, 100-15% B; 22.6-25 min,
CH:FLOW_GRADIENT                 	15% B
CH:FLOW_RATE                     	0-22.5 min, 0.333 ml/min; 22.5-22.6 min, 0.333-0.5 ml/min; 22.6-24.9 min, 0.5
CH:FLOW_RATE                     	ml/min; 24.9-25 min, 0.5-0.333 ml/min
CH:COLUMN_TEMPERATURE            	65
CH:INJECTION_TEMPERATURE         	20
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	Wistar Institute Proteomics & Metabolomic Core
AN:OPERATOR_NAME                 	Dzmitry Mukha
AN:DETECTOR_TYPE                 	Orbitrap
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive HF-X Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The HESI ion source voltage was set to the following parameters: +3,300/-3,500
MS:MS_COMMENTS                   	V, sheath gas 50, auxiliary gas 20, spare gas 0, probe heater 350 °C, capillary
MS:MS_COMMENTS                   	temperature 350 °C, S-Lens RF level 80. The mass spectrometer was set to
MS:MS_COMMENTS                   	acquire data in polarity-switching mode, one microscan, 60,000 resolution, AGC
MS:MS_COMMENTS                   	target 5e6, scan range 130-1950 m/z, IT 200 ms. Data were converted into the
MS:MS_COMMENTS                   	mzXML format by ProteoWizard and analyzed in MAVEN. Free cholesterol was
MS:MS_COMMENTS                   	measured as a water-loss in-source fragment [M-H2O+H]+.
MS:CAPILLARY_TEMPERATURE         	350
MS:ION_SOURCE_TEMPERATURE        	350
MS:ION_SPRAY_VOLTAGE             	3300
MS:IONIZATION                    	Both
MS:SOURCE_TEMPERATURE            	350
MS:AUTOMATIC_GAIN_CONTROL        	5e6
MS:DATAFORMAT                    	RAW
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	AU
MS_METABOLITE_DATA_START
Samples	01_FFA_Blank	02_FFA_Blank	03_FFA_Blank	04_FFA_D2O_BT474_Control	05_FFA_D2O_BT474_GSK0001	06_FFA_D2O_BT474_GSK0040	07_FFA_D2O_BT474_GSK1000	08_FFA_D2O_BT474_Fasnall01	09_FFA_D2O_BT474_Fasnall05	10_FFA_D2O_BT474_Fasnall40	11_FFA_D2O_BT474_Control	12_FFA_D2O_BT474_GSK0001	13_FFA_D2O_BT474_GSK0040	14_FFA_D2O_BT474_GSK1000	15_FFA_D2O_BT474_Fasnall01	16_FFA_D2O_BT474_Fasnall05	17_FFA_D2O_BT474_Fasnall40	18_FFA_D2O_BT474_Control	19_FFA_D2O_BT474_GSK0001	20_FFA_D2O_BT474_GSK0040	21_FFA_D2O_BT474_GSK1000	22_FFA_D2O_BT474_Fasnall01	23_FFA_D2O_BT474_Fasnall05	24_FFA_D2O_BT474_Fasnall40	25_FFA_Blank	26_FFA_Blank	27_FFA_Blank
Factors	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:Vehicle	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 nM GSK2194069	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 nM GSK2194069	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM GSK2194069	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM Fasnall	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:5 uM Fasnall	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 uM Fasnall	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:Vehicle	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 nM GSK2194069	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 nM GSK2194069	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM GSK2194069	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM Fasnall	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:5 uM Fasnall	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 uM Fasnall	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:Vehicle	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 nM GSK2194069	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 nM GSK2194069	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM GSK2194069	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:1 uM Fasnall	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:5 uM Fasnall	Sample source:BT-474 cell lipid extract | Isotopic Tracer:20% D2O | Drug Treatment:40 uM Fasnall	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA	Sample source:BT-474 cell lipid extract | Isotopic Tracer:NA | Drug Treatment:NA
Cholesterol [M-H2O+H]+ C12 PARENT	219599.3	0	0	3.76E+07	3.31E+07	2.89E+07	2.93E+07	3.47E+07	3.40E+07	3.20E+07	3.48E+07	3.72E+07	3.45E+07	3.22E+07	3.37E+07	3.58E+07	2.82E+07	3.84E+07	3.55E+07	3.54E+07	3.32E+07	3.58E+07	3.27E+07	3.29E+07	0	0	0
Cholesterol [M-H2O+H]+ D-label-1	0	0	0	1461776	0	957827.6	0	1107391	0	0	0	0	1344681	3624670	0	0	3248171	0	2771207	0	0	0	1016960	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-3	0	0	0	686256.8	558953	566677.4	261738.5	189877.1	0	0	463400	596418.3	500893.9	222997.2	48249.73	0	0	644784.9	358254.1	491191.2	156446.7	122504	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-4	0	0	0	735378.8	757760.8	554551.3	373458.5	164870.1	0	0	592720.7	554694.9	596485.7	336700.8	131206.9	0	0	766578.2	621389.9	483282.4	322978.2	143735.9	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-5	0	0	0	692097.6	615402.6	577390.8	408962	0	0	0	609620.3	596673.2	629561.3	457038.9	0	0	0	734367	624043.6	523758.5	298584.2	78852.22	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-6	0	0	0	426119.1	377336.4	358373.4	214659	55350.31	0	0	443308.8	348883.4	294487.3	236783	46024.92	0	0	501254.6	339079.7	426032	206776.3	40728.71	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-7	0	0	0	269157.9	212773.9	203311.5	137529.2	46256.77	0	0	278165.8	205565.3	242193.7	174173.3	15728.8	0	0	314394.8	261387.9	199616.6	109320	43001.59	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-8	0	0	0	120342.1	104220.4	70812.5	95681.99	0	0	0	148832.1	81070.76	90043	83404.91	0	0	0	143828.6	123071.9	76485.27	58079.84	14782.26	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-9	0	0	0	17746.07	0	37507.07	0	0	0	0	39280.68	17922.64	17333.82	0	0	0	0	33246.61	16096.32	18425.38	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-10	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-11	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-12	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-13	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-14	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-15	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-16	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-17	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-18	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-19	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-20	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-21	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-22	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-23	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-24	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-25	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-26	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-27	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-28	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-29	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-30	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-31	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-32	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-33	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-34	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-35	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-36	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-37	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-38	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-39	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-40	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-41	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-42	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-43	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Cholesterol [M-H2O+H]+ D-label-44	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	retention time	quantified m/z	parent	pubchem id	kegg id	regno	comments
Cholesterol [M-H2O+H]+ C12 PARENT	9.813006	369.3522	369.3522	5997	C00187	34361
Cholesterol [M-H2O+H]+ D-label-1	9.802023	370.3566	369.3522	5997	C00187	34361
Cholesterol [M-H2O+H]+ D-label-2	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-3	9.790095	372.3711	369.3522	5997	C00187	34361
Cholesterol [M-H2O+H]+ D-label-4	9.788138	373.377	369.3522	5997	C00187	34361
Cholesterol [M-H2O+H]+ D-label-5	9.786216	374.3819	369.3522	5997	C00187	34361
Cholesterol [M-H2O+H]+ D-label-6	9.783252	375.3893	369.3522	5997	C00187	34361
Cholesterol [M-H2O+H]+ D-label-7	9.777384	376.3953	369.3522	5997	C00187	34361
Cholesterol [M-H2O+H]+ D-label-8	9.779448	377.4012	369.3522	5997	C00187	34361
Cholesterol [M-H2O+H]+ D-label-9	9.77599	378.4075	369.3522	5997	C00187	34361
Cholesterol [M-H2O+H]+ D-label-10	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-11	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-12	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-13	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-14	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-15	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-16	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-17	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-18	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-19	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-20	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-21	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-22	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-23	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-24	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-25	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-26	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-27	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-28	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-29	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-30	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-31	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-32	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-33	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-34	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-35	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-36	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-37	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-38	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-39	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-40	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-41	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-42	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-43	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
Cholesterol [M-H2O+H]+ D-label-44	NA	NA	369.3522	5997	C00187	34361	Peak was not detected
METABOLITES_END
#END