#METABOLOMICS WORKBENCH jgengatharan_20240730_132509 DATATRACK_ID:5053 STUDY_ID:ST003371 ANALYSIS_ID:AN005523 PROJECT_ID:PR002085
VERSION             	1
CREATED_ON             	July 30, 2024, 1:52 pm
#PROJECT
PR:PROJECT_TITLE                 	Altered sphingolipid biosynthetic flux and lipoprotein trafficking contribute to
PR:PROJECT_TITLE                 	trans fat-induced atherosclerosis
PR:PROJECT_SUMMARY               	The goal of the project is to determine the role of sphingolipid metabolism in
PR:PROJECT_SUMMARY               	atherosclerosis induced by dietary trans fat. We analyzed lipid metabolites in
PR:PROJECT_SUMMARY               	Huh7 cells following various fatty acid treatments, with specific focus on cis
PR:PROJECT_SUMMARY               	and trans unsaturated fatty acids. Additionally, we analyzed lipid metabolites
PR:PROJECT_SUMMARY               	in plasma and liver of Ldlr-/- mice fed high-fat diets enriched in cis or trans
PR:PROJECT_SUMMARY               	fatty acids in the presence or absence of myriocin, a pharmacological inhibitor
PR:PROJECT_SUMMARY               	of SPT, the initial rate-limiting enzyme of sphingolipid biosynthesis.
PR:INSTITUTE                     	Salk Institute for Biological Studies
PR:LAST_NAME                     	Gengatharan
PR:FIRST_NAME                    	Jivani
PR:ADDRESS                       	10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA
PR:EMAIL                         	jivani14@gmail.com
PR:PHONE                         	(858) 453-4100
#STUDY
ST:STUDY_TITLE                   	Incorporation of oleate-d9 and elaidate-d17 in broader lipidome of Huh7 cells.
ST:STUDY_SUMMARY                 	We analyzed several lipid classes including LPC, LPE, PC, PE, DG, and TG in Huh7
ST:STUDY_SUMMARY                 	cells treated with BSA-oleate-d9 or BSA-elaidate-d17 to determine their
ST:STUDY_SUMMARY                 	incorporation into the broader lipidome.
ST:INSTITUTE                     	Salk Institute for Biological Studies
ST:LAST_NAME                     	Gengatharan
ST:FIRST_NAME                    	Jivani
ST:ADDRESS                       	10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA
ST:EMAIL                         	jivani14@gmail.com
ST:PHONE                         	(858) 453-4100
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:CELL_STRAIN_DETAILS           	Huh7
#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           	-	Huh7_C30Lip1_oleicd9_1	Sample source:Huh7 cells | Treatment:BSA-oleate-d9	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_oleicd9_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C30Lip1_oleicd9_2	Sample source:Huh7 cells | Treatment:BSA-oleate-d9	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_oleicd9_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C30Lip1_oleicd9_3	Sample source:Huh7 cells | Treatment:BSA-oleate-d9	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_oleicd9_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C30Lip1_elaidicd17_1	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_elaidicd17_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C30Lip1_elaidicd17_2	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_elaidicd17_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C30Lip1_elaidicd17_3	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_elaidicd17_3.d
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells (~400,000 cells)were spiked with 1 µg of each of the following internal
CO:COLLECTION_SUMMARY            	standards: 15:0-18:1(d7) phosphatidylcholine (Avanti Polar Lipids, Cat #791637),
CO:COLLECTION_SUMMARY            	15:0-18:1(d7) phosphatidylethanolamine (Avanti Polar Lipids, Cat #791638),
CO:COLLECTION_SUMMARY            	18:1(d7) lysophosphatidylcholine (Avanti Polar Lipids, Cat#791643), 18:1(d7)
CO:COLLECTION_SUMMARY            	lysophosphatidylethanolamine (Avanti Polar Lipids, Cat #791644), 15:0-18:1(d7)
CO:COLLECTION_SUMMARY            	diacylglycerol (Avanti Polar Lipids, Cat #791647), 15:0-18:1(d7)-15:0
CO:COLLECTION_SUMMARY            	triacylglycerol (Avanti Polar Lipids, Cat #791648). Cells were scraped with 0.5
CO:COLLECTION_SUMMARY            	mL methanol and 0.5 mL H2O.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Huh7 cells were treated with 1) 100 µM BSA-oleate-d9 or 2) 100 µM
TR:TREATMENT_SUMMARY             	BSA-elaidate-d17 for 48 hours in delipidated media.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Cells (~400,000 cells) were spiked with 1 µg of each of the following internal
SP:SAMPLEPREP_SUMMARY            	standards: 15:0-18:1(d7) phosphatidylcholine (Avanti Polar Lipids, Cat #791637),
SP:SAMPLEPREP_SUMMARY            	15:0-18:1(d7) phosphatidylethanolamine (Avanti Polar Lipids, Cat #791638),
SP:SAMPLEPREP_SUMMARY            	18:1(d7) lysophosphatidylcholine (Avanti Polar Lipids, Cat#791643), 18:1(d7)
SP:SAMPLEPREP_SUMMARY            	lysophosphatidylethanolamine (Avanti Polar Lipids, Cat #791644), 15:0-18:1(d7)
SP:SAMPLEPREP_SUMMARY            	diacylglycerol (Avanti Polar Lipids, Cat #791647), 15:0-18:1(d7)-15:0
SP:SAMPLEPREP_SUMMARY            	triacylglycerol (Avanti Polar Lipids, Cat #791648). Cells were scraped with 0.5
SP:SAMPLEPREP_SUMMARY            	mL methanol and 0.5 mL H2O. Homogenate aliquot of 100 µL was taken to determine
SP:SAMPLEPREP_SUMMARY            	protein content using the BCA protein assay (Thermo Fisher Scientific). The
SP:SAMPLEPREP_SUMMARY            	remaining homogenate was transferred to a new Eppendorf tube and 1 mL chloroform
SP:SAMPLEPREP_SUMMARY            	was added. For media, 0.5 mL methanol, 0.5 mL H2O, and 1 mL chloroform were
SP:SAMPLEPREP_SUMMARY            	added directly. Samples were vortexed for 5 min and centrifuged for 5 min at 4
SP:SAMPLEPREP_SUMMARY            	˚C at 15,000g. The organic phase was collected and 2 μL of formic acid was
SP:SAMPLEPREP_SUMMARY            	added to the remaining polar phase which was re-extracted with 1 mL of
SP:SAMPLEPREP_SUMMARY            	chloroform. Combined organic phases were dried under nitrogen. After dried
SP:SAMPLEPREP_SUMMARY            	extracts were resuspended in 60 µl 65:30:5 ACN: IPA:H2O, 5 µL of sample was
SP:SAMPLEPREP_SUMMARY            	injected.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1260 Infinity II
CH:COLUMN_NAME                   	Thermo Accucore C30 (150 x 2.1mm,2.6um)
CH:SOLVENT_A                     	60% acetonitrile, 40% water; 0.1% formic acid; 10 mM ammonium formate
CH:SOLVENT_B                     	90% isopropanol, 10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
CH:FLOW_GRADIENT                 	0 min, 30% B; 3 min, 30% B; 8 min, 43% B; 9 min, 50% B; 18 min, 90% B; 26 min,
CH:FLOW_GRADIENT                 	99% B; 30 min, 99%B; 36 min, 30% B
CH:FLOW_RATE                     	0 min, 0.1 ml/min; 8 min, 0.2 ml/min
CH:COLUMN_TEMPERATURE            	40˚C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6460 QQQ
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Lipid species were analyzed by multiple reaction monitoring of the transition
MS:MS_COMMENTS                   	from precursor to product ions at associated optimized collision energies, and
MS:MS_COMMENTS                   	fragmentor voltages using Agilent Masshunter. The m/z values of the precursor
MS:MS_COMMENTS                   	and product ions are provided in the metabolite metadata section.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Peak area
MS_METABOLITE_DATA_START
Samples	Huh7_C30Lip1_oleicd9_1	Huh7_C30Lip1_oleicd9_2	Huh7_C30Lip1_oleicd9_3	Huh7_C30Lip1_elaidicd17_1	Huh7_C30Lip1_elaidicd17_2	Huh7_C30Lip1_elaidicd17_3
Factors	Sample source:Huh7 cells | Treatment:BSA-oleate-d9	Sample source:Huh7 cells | Treatment:BSA-oleate-d9	Sample source:Huh7 cells | Treatment:BSA-oleate-d9	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17
LPC 18:1-d7	9007710	7871056	6511425	8186781	7278011	8064780
LPC 16:1-d17	0	0	0	340428	330371	318885
LPC 18:1-d17	0	0	0	2516108	2729106	2546011
LPC 18:1-d9	1606944	1689914	2223580	0	0	0
LPC 20:1-d9	12600	13421	18290	0	0	0
LPE 18:1-d7	978294	996634	624139	1001338	919329	936759
LPE 18:1-d17	0	0	0	114365	115650	117027
LPE 18:1-d9	49129	46689	30287	0	0	0
PC 15:0/18:1-d7	51162298	49584456	39608898	31516879	36170360	34235427
PC 32:1-d17	0	0	0	14002965	16256387	15122397
PC 32:2-d34	0	0	0	14739601	16220445	14765212
PC 32:1-d9	17210397	18691827	14855864	0	0	0
PC 32:2-d17	0	0	0	11229639	11777252	9503720
PC 34:1-d17	0	0	0	14937219	17961852	16745976
PC 34:1-d9	70705229	69489912	61454435	0	0	0
PC 34:2-d17	0	0	0	21493093	40514370	39985740
PC 34:2-d18	30265723	29254741	23556344	0	0	0
PC 34:2-d34	0	0	0	26386628	30376560	27406364
PC 34:2-d9	36829616	38415763	32327874	0	0	0
PC 36:1-d9	65164589	65287160	43688784	0	0	0
PC 36:2-d17	0	0	0	29078897	31826370	26972435
PC 36:2-d18	111143069	102962279	101095554	0	0	0
PC 36:2-d34	0	0	0	51531300	55858590	56737131
PC 36:2-d9	39047168	37592611	32400856	0	0	0
PC 36:3-d17	0	0	0	11264850	11467339	12963183
PC 36:3-d9	9693402	12221271	9642547	0	0	0
PE 15:0/18:1-d7	61768	67153	64822	126971	41889	159401
PE 34:2-d17	0	0	0	31132	33288	34801
PE 36:1-d9	18314	71102	11001	0	0	0
PE 36:2-d17	0	0	0	23273	53874	43313
PE 36:2-d18	181422	405830	294478	0	0	0
PE 36:2-d9	25137	26609	33944	0	0	0
PE 36:3-d17	0	0	0	27558	11133	30098
PE 38:2-d18	92098	112859	25112	0	0	0
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Precursor Ion	Product Ion
LPC 18:1-d7	529.4	184.1
LPC 16:1-d17	511.3	184.1
LPC 18:1-d17	539.3	184.1
LPC 18:1-d9	531.3	184.1
LPC 20:1-d9	559.3	184.1
LPE 18:1-d7	487.3	44.1
LPE 18:1-d17	497.3	44.1
LPE 18:1-d9	489.3	44.1
PC 15:0/18:1-d7	753.6	184.1
PC 32:1-d17	749.6	184.1
PC 32:2-d34	764.6	184.1
PC 32:1-d9	741.6	184.1
PC 32:2-d17	747.6	184.1
PC 34:1-d17	777.6	184.1
PC 34:1-d9	769.6	184.1
PC 34:2-d17	775.6	184.1
PC 34:2-d18	776.6	184.1
PC 34:2-d34	792.6	184.1
PC 34:2-d9	767.6	184.1
PC 36:1-d9	797.6	184.1
PC 36:2-d17	803.6	184.1
PC 36:2-d18	804.6	184.1
PC 36:2-d34	820.6	184.1
PC 36:2-d9	795.6	184.1
PC 36:3-d17	801.6	184.1
PC 36:3-d9	793.6	184.1
PE 15:0/18:1-d7	711.6	570.5
PE 34:2-d17	733.5	592.5
PE 36:1-d9	755.6	614.5
PE 36:2-d17	761.6	620.5
PE 36:2-d18	762.6	621.5
PE 36:2-d9	753.6	612.5
PE 36:3-d17	759.5	618.5
PE 38:2-d18	790.6	649.5
METABOLITES_END
#END