#METABOLOMICS WORKBENCH jgengatharan_20240730_162438 DATATRACK_ID:5060 STUDY_ID:ST003360 ANALYSIS_ID:AN005502 PROJECT_ID:PR002085
VERSION             	1
CREATED_ON             	July 30, 2024, 4:31 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                   	Secretion of sphingomyelin from Huh7 cells treated with oleate-d9 or
ST:STUDY_TITLE                   	elaidate-d17 while modulating SPT flux.
ST:STUDY_SUMMARY                 	We analyzed SM in the fresh and spent media from Huh7 cells treated with
ST:STUDY_SUMMARY                 	BSA-oleate-d9 or BSA-elaidate-d17. We validated their reduction in abundance
ST:STUDY_SUMMARY                 	through pharmacological inhibition of SPT with myriocin.
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           	-	H7freshmed_delip_1	Sample source:Huh7 media | Condition:Fresh media	RAW_FILE_NAME(Raw file name)=H7freshmed_delip_1.d
SUBJECT_SAMPLE_FACTORS           	-	H7freshmed_delip_2	Sample source:Huh7 media | Condition:Fresh media	RAW_FILE_NAME(Raw file name)=H7freshmed_delip_2.d
SUBJECT_SAMPLE_FACTORS           	-	H7freshmed_delip_3	Sample source:Huh7 media | Condition:Fresh media	RAW_FILE_NAME(Raw file name)=H7freshmed_delip_3.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Od9_1	Sample source:Huh7 media | Condition:BSA-oleate-d9 + DMSO spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Od9_1.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Od9_2	Sample source:Huh7 media | Condition:BSA-oleate-d9 + DMSO spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Od9_2.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Od9_3	Sample source:Huh7 media | Condition:BSA-oleate-d9 + DMSO spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Od9_3.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Od9myr_1	Sample source:Huh7 media | Condition:BSA-oleate-d9 + myriocin spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Od9myr_1.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Od9myr_2	Sample source:Huh7 media | Condition:BSA-oleate-d9 + myriocin spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Od9myr_2.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Od9myr_3	Sample source:Huh7 media | Condition:BSA-oleate-d9 + myriocin spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Od9myr_3.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Ed17_1	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + DMSO spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Ed17_1.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Ed17_2	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + DMSO spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Ed17_2.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Ed17_3	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + DMSO spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Ed17_3.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Ed17myr_1	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + myriocin spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Ed17myr_1.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Ed17myr_2	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + myriocin spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Ed17myr_2.d
SUBJECT_SAMPLE_FACTORS           	-	H7spentmed_delip_Ed17myr_3	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + myriocin spent media	RAW_FILE_NAME(Raw file name)=H7spentmed_delip_Ed17myr_3.d
#COLLECTION
CO:COLLECTION_SUMMARY            	0.75 ml of fresh media or 1.5 ml of spent media was evaporated under vacuum at
CO:COLLECTION_SUMMARY            	4°C and resuspended into 0.1 mL of H2O.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Huh7 cells were treated with 1) 100 µM BSA-oleate-d9 with DMSO, 2) 100 µM
TR:TREATMENT_SUMMARY             	BSA-100 µM oleate-d9 with 100 nM myriocin, 3) 100 µM BSA-elaidate-d17 with
TR:TREATMENT_SUMMARY             	DMSO, or 4) 100 µM BSA-elaidate-d17 with 100 nM myriocin for 48 hours in
TR:TREATMENT_SUMMARY             	delipidated media.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Media was spiked with the following internal standards: 20 pmol sphinganine-d7
SP:SAMPLEPREP_SUMMARY            	(Avanti Polar Lipids, Cat# 860658), deoxysphinganine-d3 (Avanti Polar Lipids,
SP:SAMPLEPREP_SUMMARY            	Cat# 860474), 100 pmol d18:0-d7/13:0 dihydroceramide (Avanti Polar Lipids, Cat#
SP:SAMPLEPREP_SUMMARY            	330726), 200 pmol d18:1-d7/15:0 ceramide (Avanti Polar Lipids, Cat# 860681), 100
SP:SAMPLEPREP_SUMMARY            	pmol d18:1-d7/15:0 glucosylceramide (Avanti Polar Lipids, Cat# 330729), 100 pmol
SP:SAMPLEPREP_SUMMARY            	d18:1-d7/15:0 lactosylceramide (Avanti Polar Lipids, Cat# 330727), 200 pmol
SP:SAMPLEPREP_SUMMARY            	sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), 200 pmol sphingosine-d7
SP:SAMPLEPREP_SUMMARY            	(Avanti Polar Lipids, Cat# 860657), and 200 pmol sphingomyelin (d18:1/18:1)-d9
SP:SAMPLEPREP_SUMMARY            	(Avanti Polar Lipids, Cat#860740). 0.5 mL methanol, 0.5 mL H2O, and 1 mL
SP:SAMPLEPREP_SUMMARY            	chloroform were added directly. Samples were vortexed for 5 min and centrifuged
SP:SAMPLEPREP_SUMMARY            	for 5 min at 4 ˚C at 15,000g. The organic phase was collected and 2 μL of
SP:SAMPLEPREP_SUMMARY            	formic acid was added to the remaining polar phase which was re-extracted with 1
SP:SAMPLEPREP_SUMMARY            	mL of chloroform. Combined organic phases were dried under nitrogen. After dried
SP:SAMPLEPREP_SUMMARY            	extracts for cells were resuspended in 60 µl Buffer B, 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                   	Peeke Scientific Spectra C8SR (150 x 3.0 mm, 3μm)
CH:SOLVENT_A                     	100% water; 0.2% formic acid; 2 mM ammonium formate
CH:SOLVENT_B                     	100% methanol; 0.2% formic acid; 1 mM ammonium formate
CH:FLOW_GRADIENT                 	0 min, 82% B; 3 min, 82% B; 4 min, 90% B; 18 min, 99% B; 25 min, 99% B; 27 min,
CH:FLOW_GRADIENT                 	82% B; 30 min, 82% B
CH:FLOW_RATE                     	0.5 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                   	Sphingolipid species were analyzed by multiple reaction monitoring of the
MS:MS_COMMENTS                   	transition from precursor to product ions at associated optimized collision
MS:MS_COMMENTS                   	energies, and fragmentor voltages using Agilent Masshunter. The m/z values of
MS:MS_COMMENTS                   	the precursor and product ions are provided in the metabolite metadata section.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Peak area
MS_METABOLITE_DATA_START
Samples	H7freshmed_delip_1	H7freshmed_delip_2	H7freshmed_delip_3	H7spentmed_delip_Od9_1	H7spentmed_delip_Od9_2	H7spentmed_delip_Od9_3	H7spentmed_delip_Od9myr_1	H7spentmed_delip_Od9myr_2	H7spentmed_delip_Od9myr_3	H7spentmed_delip_Ed17_1	H7spentmed_delip_Ed17_2	H7spentmed_delip_Ed17_3	H7spentmed_delip_Ed17myr_1	H7spentmed_delip_Ed17myr_2	H7spentmed_delip_Ed17myr_3
Factors	Sample source:Huh7 media | Condition:Fresh media	Sample source:Huh7 media | Condition:Fresh media	Sample source:Huh7 media | Condition:Fresh media	Sample source:Huh7 media | Condition:BSA-oleate-d9 + DMSO spent media	Sample source:Huh7 media | Condition:BSA-oleate-d9 + DMSO spent media	Sample source:Huh7 media | Condition:BSA-oleate-d9 + DMSO spent media	Sample source:Huh7 media | Condition:BSA-oleate-d9 + myriocin spent media	Sample source:Huh7 media | Condition:BSA-oleate-d9 + myriocin spent media	Sample source:Huh7 media | Condition:BSA-oleate-d9 + myriocin spent media	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + DMSO spent media	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + DMSO spent media	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + DMSO spent media	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + myriocin spent media	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + myriocin spent media	Sample source:Huh7 media | Condition:BSA-elaidate-d17 + myriocin spent media
SM (d18:1/18:1)-d9	2054351	1616464	1497012	1484726	1447235	2240363	2746257	3814232	3887066	4097672	1990072	2280692	1657489	2236493	2109087
SM d32:2-d17	0	0	0	0	0	0	0	0	0	149416	126174	137319	57369	53997	68575
SM d32:2-d9	0	0	0	30538	22870	35792	7403	3435	3094	0	0	0	0	0	0
SM d34:2-d17	0	0	0	0	0	0	0	0	0	2030385	1645417	1736167	828917	538927	467658
SM d34:2-d9	0	0	0	562123	499159	500050	52135	59860	51454	0	0	0	0	0	0
SM d34:3-d17	0	0	0	0	0	0	0	0	0	153847	148011	165793	149341	149141	88452
SM d36:2-d17	0	0	0	0	0	0	0	0	0	456422	325483	381709	454098	200139	186069
SM d36:2-d34	0	0	0	0	0	0	0	0	0	320986	243994	304513	41836	29784	27797
SM d36:2-d9	0	0	0	1328139	1272458	1506386	266435	302993	258422	0	0	0	0	0	0
SM d36:3-d17	0	0	0	0	0	0	0	0	0	280378	214192	230343	337020	145149	125191
SM d36:3-d34	0	0	0	0	0	0	0	0	0	4782699	3718975	3667393	167037	151090	113010
SM d36:3-d9	0	0	0	153013	210682	195775	95357	60186	36429	0	0	0	0	0	0
SM d38:3-d34	0	0	0	0	0	0	0	0	0	486608	122327	197081	188585	115249	49793
SM d40:3-d34	0	0	0	0	0	0	0	0	0	144539	84572	110993	57450	40813	44558
SM d42:3-d17	0	0	0	0	0	0	0	0	0	38981	38525	37066	7637	7066	7077
SM d42:3-d34	0	0	0	0	0	0	0	0	0	198223	119649	118584	33247	12244	15908
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Precursor Ion	Product Ion
SM (d18:1/18:1)-d9	738.6	193
SM d32:2-d17	690.5	184
SM d32:2-d9	682.5	184
SM d34:2-d17	718.6	184
SM d34:2-d9	710.6	184
SM d34:3-d17	716.5	184
SM d36:2-d17	746.6	184
SM d36:2-d34	763.6	184
SM d36:2-d9	738.6	184
SM d36:3-d17	744.6	184
SM d36:3-d34	761.6	184
SM d36:3-d9	736.6	184
SM d38:3-d34	789.6	184
SM d40:3-d34	817.6	184
SM d42:3-d17	828.7	184
SM d42:3-d34	845.7	184
METABOLITES_END
#END