#METABOLOMICS WORKBENCH jgengatharan_20240730_142213 DATATRACK_ID:5057 STUDY_ID:ST003373 ANALYSIS_ID:AN005525 PROJECT_ID:PR002085
VERSION             	1
CREATED_ON             	July 30, 2024, 2:29 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 sphingolipids in Huh7 cells while
ST:STUDY_TITLE                   	modulating SPT flux.
ST:STUDY_SUMMARY                 	We analyzed sphingolipids in Huh7 cells treated with BSA-oleate-d9 or
ST:STUDY_SUMMARY                 	BSA-elaidate-d17 to determine their incorporation into the long-chain base (LCB)
ST:STUDY_SUMMARY                 	of intact sphingolipids. We validated their reduction in abundance through
ST:STUDY_SUMMARY                 	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           	-	Huh7_delip_SL1_Od9_1	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_delip_SL1_Od9_2	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_delip_SL1_Od9_3	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_delip_SL1_Od9myr_1	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9myr_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_delip_SL1_Od9myr_2	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9myr_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_delip_SL1_Od9myr_3	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9myr_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_delip_SL1_Ed17_1	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Ed17_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_delip_SL1_Ed17_2	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Ed17_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_delip_SL1_Ed17_3	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Ed17_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_delip_SL1_Ed17myr_1	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Ed17myr_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_delip_SL1_Ed17myr_2	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Ed17myr_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_delip_SL1_Ed17myr_3	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin	RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Ed17myr_3.d
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells (~400,000 cells) were spiked with the following internal standards: 20
CO:COLLECTION_SUMMARY            	pmol sphinganine-d7 (Avanti Polar Lipids, Cat# 860658), deoxysphinganine-d3
CO:COLLECTION_SUMMARY            	(Avanti Polar Lipids, Cat# 860474), 100 pmol d18:0-d7/13:0 dihydroceramide
CO:COLLECTION_SUMMARY            	(Avanti Polar Lipids, Cat# 330726), 200 pmol d18:1-d7/15:0 ceramide (Avanti
CO:COLLECTION_SUMMARY            	Polar Lipids, Cat# 860681), 100 pmol d18:1-d7/15:0 glucosylceramide (Avanti
CO:COLLECTION_SUMMARY            	Polar Lipids, Cat# 330729), 100 pmol d18:1-d7/15:0 lactosylceramide (Avanti
CO:COLLECTION_SUMMARY            	Polar Lipids, Cat# 330727), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat#
CO:COLLECTION_SUMMARY            	860657), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), and 200
CO:COLLECTION_SUMMARY            	pmol d18:1/18:1-d9 sphingomyelin (Avanti Polar Lipids, Cat# 791649) or 200 pmol
CO:COLLECTION_SUMMARY            	sphingomyelin (d18:1/18:1)-d9 (Avanti Polar Lipids, Cat#860740). Cells were
CO:COLLECTION_SUMMARY            	scraped with 0.5 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 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            	Cells (~400,000 cells) were spiked with the following internal standards: 20
SP:SAMPLEPREP_SUMMARY            	pmol sphinganine-d7 (Avanti Polar Lipids, Cat# 860658), deoxysphinganine-d3
SP:SAMPLEPREP_SUMMARY            	(Avanti Polar Lipids, Cat# 860474), 100 pmol d18:0-d7/13:0 dihydroceramide
SP:SAMPLEPREP_SUMMARY            	(Avanti Polar Lipids, Cat# 330726), 200 pmol d18:1-d7/15:0 ceramide (Avanti
SP:SAMPLEPREP_SUMMARY            	Polar Lipids, Cat# 860681), 100 pmol d18:1-d7/15:0 glucosylceramide (Avanti
SP:SAMPLEPREP_SUMMARY            	Polar Lipids, Cat# 330729), 100 pmol d18:1-d7/15:0 lactosylceramide (Avanti
SP:SAMPLEPREP_SUMMARY            	Polar Lipids, Cat# 330727), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat#
SP:SAMPLEPREP_SUMMARY            	860657), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), and 200
SP:SAMPLEPREP_SUMMARY            	pmol d18:1/18:1-d9 sphingomyelin (Avanti Polar Lipids, Cat# 791649) or 200 pmol
SP:SAMPLEPREP_SUMMARY            	sphingomyelin (d18:1/18:1)-d9 (Avanti Polar Lipids, Cat#860740). Cells were
SP:SAMPLEPREP_SUMMARY            	scraped with 0.5 mL methanol and 0.5 mL H2O. Homogenate aliquot of 100 µL was
SP:SAMPLEPREP_SUMMARY            	taken to determine protein content using the BCA protein assay (Thermo Fisher
SP:SAMPLEPREP_SUMMARY            	Scientific). The remaining homogenate was transferred to a new Eppendorf tube
SP:SAMPLEPREP_SUMMARY            	and 1 mL chloroform was added. For plasma or media, 0.5 mL methanol, 0.5 mL H2O,
SP:SAMPLEPREP_SUMMARY            	and 1 mL chloroform were added directly. Samples were vortexed for 5 min and
SP:SAMPLEPREP_SUMMARY            	centrifuged for 5 min at 4 ˚C at 15,000g. The organic phase was collected and 2
SP:SAMPLEPREP_SUMMARY            	μL of formic acid was added to the remaining polar phase which was re-extracted
SP:SAMPLEPREP_SUMMARY            	with 1 mL of chloroform. Combined organic phases were dried under nitrogen.
SP:SAMPLEPREP_SUMMARY            	After dried extracts for cells were resuspended in 60 µl Buffer B, 5 µL of
SP:SAMPLEPREP_SUMMARY            	sample was 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	Huh7_delip_SL1_Od9_1	Huh7_delip_SL1_Od9_2	Huh7_delip_SL1_Od9_3	Huh7_delip_SL1_Od9myr_1	Huh7_delip_SL1_Od9myr_2	Huh7_delip_SL1_Od9myr_3	Huh7_delip_SL1_Ed17_1	Huh7_delip_SL1_Ed17_2	Huh7_delip_SL1_Ed17_3	Huh7_delip_SL1_Ed17myr_1	Huh7_delip_SL1_Ed17myr_2	Huh7_delip_SL1_Ed17myr_3	
Factors	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin	Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin	Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin
Cer d18:1-d7/15:0	2005739	1690413	1820713	2652621	2580746	2898124	1746168	2076586	1864612	2096203	2464482	2661620	
Cer d20:2-d17/18:1-d17							3574	1988	3818				
Cer d20:2-d17/22:0							4400	2075	1940				
Cer d20:2-d17/22:1							937	2618	1545				
Cer d20:2-d17/22:1-d17							3146	3890	3968				
Cer d20:2-d17/24:0							3224	5980	4157				
Cer d20:2-d17/24:1							5770	4878	6807				
Cer d20:2-d17/24:1-d17							8905	7999	7836				
Gluc-Cer d18:1-d7/15:0	395013	345502	370367	224477	359383	352413	360433	469754	442026	459585	422974	428587	
Hex-Cer d20:2-d17/22:1-d17							2121	864	1226				
Hex-Cer d20:2-d17/24:1-d17							904	1226	1366				
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Precursor Ion	Product Ion
Cer d18:1-d7/15:0	531.5	271.4
Cer d20:2-d17/18:1-d17	624.6	307.4
Cer d20:2-d17/22:0	665.6	307.4
Cer d20:2-d17/22:1	663.6	307.4
Cer d20:2-d17/22:1-d17	680.6	307.4
Cer d20:2-d17/24:0	693.6	307.4
Cer d20:2-d17/24:1	691.6	307.4
Cer d20:2-d17/24:1-d17	708.6	307.4
Gluc-Cer d18:1-d7/15:0	693.6	271.2
Hex-Cer d20:2-d17/22:1-d17	842.7	307.4
Hex-Cer d20:2-d17/24:1-d17	870.7	307.4
METABOLITES_END
#END