#METABOLOMICS WORKBENCH jgengatharan_20240730_135434 DATATRACK_ID:5055 STUDY_ID:ST003372 ANALYSIS_ID:AN005524 PROJECT_ID:PR002085
VERSION             	1
CREATED_ON             	July 30, 2024, 1:58 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_C30Lip2_oleicd9_1	Sample source:Huh7 cells | Treatment:BSA-oleate-d9	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_oleicd9_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C30Lip2_oleicd9_2	Sample source:Huh7 cells | Treatment:BSA-oleate-d9	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_oleicd9_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C30Lip2_oleicd9_3	Sample source:Huh7 cells | Treatment:BSA-oleate-d9	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_oleicd9_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C30Lip2_elaidicd17_1	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_elaidicd17_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C30Lip2_elaidicd17_2	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_elaidicd17_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C30Lip2_elaidicd17_3	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17	RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_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_C30Lip2_oleicd9_1	Huh7_C30Lip2_oleicd9_2	Huh7_C30Lip2_oleicd9_3	Huh7_C30Lip2_elaidicd17_1	Huh7_C30Lip2_elaidicd17_2	Huh7_C30Lip2_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
DG 15:0/18:1-d7	30737	10728	22411	9898	21890	45925	
DG 18:1-d17_18:1	0	0	0	2736	1490	5415	
DG 18:1-d17_18:1-d17	0	0	0	20113	18407	14428	
DG 18:1-d9_18:1	12917	11206	9265	0	0	0	
DG 18:1-d9_18:1-d9	37964	29442	30177	0	0	0	
TG 15:0/18:1-d7/15:0	277268	200036	51463	52941	154999	173346	
TG 50:2-d34 (dC32:1-d17)	0	0	0	839525	1427547	813922	
TG 52:2-d18 (dC34:1-d9)	827266	879628	752650	0	0	0	
TG 52:2-d34 (dC34:1-d17)	0	0	0	84472	1093279	101593	
TG 52:3-d18 (dC34:2-d9)	614823	595128	597463	0	0	0	
TG 52:3-d27 (dC34:2-d18)	403701	403150	445166	0	0	0	
TG 52:3-d34 (dC34:2-d17)	0	0	0	835710	2288528	2271478	
TG 52:3-d51 (dC34:2-d34)	0	0	0	217848	1036289	604686	
TG 54:2-d34 (dC36:1-d17)	0	0	0	19193	574518	33673	
TG 54:3-d18 (dC36:2-d9)	770524	857878	932934	0	0	0	
TG 54:3-d27 (dC36:2-d18)	10200762	3318728	2527687	0	0	0	
TG 54:3-d34 (dC36:2-d17)	0	0	0	948031	1759145	1497614	
TG 54:3-d51 (dC36:2-d34)	0	0	0	336756	5626350	2246677	
TG 54:4-d34 (dC36:3-d17)	0	0	0	313987	374057	421849	
TG 56:2-d18 (dC38:1-d9)	659584	588723	442650	0	0	0	
TG 56:3-d27 (dC38:2-d18)	676179	654568	646440	0	0	0	
TG 56:3-d34 (dC38:2-d17)	0	0	0	645948	204624	139645	
TG 56:3-d51 (dC38:2-d34)	0	0	0	94009	188847	42967	
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Precursor Ion	Product Ion
DG 15:0/18:1-d7	605.6	299
DG 18:1-d17_18:1	655.5	356
DG 18:1-d17_18:1-d17	672.5	356
DG 18:1-d9_18:1	647.5	348
DG 18:1-d9_18:1-d9	656.5	348
TG 15:0/18:1-d7/15:0	829.8	523.5
TG 50:2-d34 (dC32:1-d17)	882.8	566.5
TG 52:2-d18 (dC34:1-d9)	894.8	586.5
TG 52:2-d34 (dC34:1-d17)	910.8	594.5
TG 52:3-d18 (dC34:2-d9)	892.8	584.5
TG 52:3-d27 (dC34:2-d18)	901.8	593.5
TG 52:3-d34 (dC34:2-d17)	908.8	592.5
TG 52:3-d51 (dC34:2-d34)	925.8	609.5
TG 54:2-d34 (dC36:1-d17)	938.9	622.6
TG 54:3-d18 (dC36:2-d9)	920.9	612.6
TG 54:3-d27 (dC36:2-d18)	929.9	621.6
TG 54:3-d34 (dC36:2-d17)	936.9	620.6
TG 54:3-d51 (dC36:2-d34)	953.9	637.6
TG 54:4-d34 (dC36:3-d17)	934.8	618.5
TG 56:2-d18 (dC38:1-d9)	950.9	642.5
TG 56:3-d27 (dC38:2-d18)	957.9	649.5
TG 56:3-d34 (dC38:2-d17)	964.9	648.5
TG 56:3-d51 (dC38:2-d34)	981.9	665.5
METABOLITES_END
#END