#METABOLOMICS WORKBENCH jgengatharan_20240725_092925 DATATRACK_ID:5033 STUDY_ID:ST003351 ANALYSIS_ID:AN005492 PROJECT_ID:PR002085
VERSION             	1
CREATED_ON             	July 26, 2024, 8:09 am
#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 various fatty acids in long-chain base of sphingolipids in Huh7
ST:STUDY_TITLE                   	cells
ST:STUDY_SUMMARY                 	We analyzed hydrolyzed long-chain bases (LCBs) in Huh7 cells treated with
ST:STUDY_SUMMARY                 	various BSA-conjugated fatty acids.
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_palm_C16_1	Sample source:Huh7 cells | Treatment:BSA-palmitate	RAW_FILE_NAME(Raw file name)=Huh7_palm_C16_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_palm_C16_2	Sample source:Huh7 cells | Treatment:BSA-palmitate	RAW_FILE_NAME(Raw file name)=Huh7_palm_C16_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_palm_C16_3	Sample source:Huh7 cells | Treatment:BSA-palmitate	RAW_FILE_NAME(Raw file name)=Huh7_palm_C16_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C17_1	Sample source:Huh7 cells | Treatment:BSA-heptadecanoate	RAW_FILE_NAME(Raw file name)=Huh7_C17_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C17_2	Sample source:Huh7 cells | Treatment:BSA-heptadecanoate	RAW_FILE_NAME(Raw file name)=Huh7_C17_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_C17_3	Sample source:Huh7 cells | Treatment:BSA-heptadecanoate	RAW_FILE_NAME(Raw file name)=Huh7_C17_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_stearic_C18_1	Sample source:Huh7 cells | Treatment:BSA-stearate	RAW_FILE_NAME(Raw file name)=Huh7_stearic_C18_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_stearic_C18_2	Sample source:Huh7 cells | Treatment:BSA-stearate	RAW_FILE_NAME(Raw file name)=Huh7_stearic_C18_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_stearic_C18_3	Sample source:Huh7 cells | Treatment:BSA-stearate	RAW_FILE_NAME(Raw file name)=Huh7_stearic_C18_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_isoC16_1	Sample source:Huh7 cells | Treatment:BSA-isoC16 0	RAW_FILE_NAME(Raw file name)=Huh7_isoC16_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_isoC16_2	Sample source:Huh7 cells | Treatment:BSA-isoC16 0	RAW_FILE_NAME(Raw file name)=Huh7_isoC16_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_isoC16_3	Sample source:Huh7 cells | Treatment:BSA-isoC16 0	RAW_FILE_NAME(Raw file name)=Huh7_isoC16_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_anteisoC17_1	Sample source:Huh7 cells | Treatment:BSA-anteisoC17 0	RAW_FILE_NAME(Raw file name)=Huh7_anteisoC17_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_anteisoC17_2	Sample source:Huh7 cells | Treatment:BSA-anteisoC17 0	RAW_FILE_NAME(Raw file name)=Huh7_anteisoC17_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_anteisoC17_3	Sample source:Huh7 cells | Treatment:BSA-anteisoC17 0	RAW_FILE_NAME(Raw file name)=Huh7_anteisoC17_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_isoC17_1	Sample source:Huh7 cells | Treatment:BSA-isoC17 0	RAW_FILE_NAME(Raw file name)=Huh7_isoC17_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_isoC17_2	Sample source:Huh7 cells | Treatment:BSA-isoC17 0	RAW_FILE_NAME(Raw file name)=Huh7_isoC17_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_isoC17_3	Sample source:Huh7 cells | Treatment:BSA-isoC17 0	RAW_FILE_NAME(Raw file name)=Huh7_isoC17_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_oleic_C18-1cis_1	Sample source:Huh7 cells | Treatment:BSA-oleate	RAW_FILE_NAME(Raw file name)=Huh7_oleic_C18-1cis_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_oleic_C18-1cis_2	Sample source:Huh7 cells | Treatment:BSA-oleate	RAW_FILE_NAME(Raw file name)=Huh7_oleic_C18-1cis_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_oleic_C18-1cis_3	Sample source:Huh7 cells | Treatment:BSA-oleate	RAW_FILE_NAME(Raw file name)=Huh7_oleic_C18-1cis_3.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_elaidic_C18-1trans_1	Sample source:Huh7 cells | Treatment:BSA-elaidate	RAW_FILE_NAME(Raw file name)=Huh7_elaidic_C18-1trans_1.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_elaidic_C18-1trans_2	Sample source:Huh7 cells | Treatment:BSA-elaidate	RAW_FILE_NAME(Raw file name)=Huh7_elaidic_C18-1trans_2.d
SUBJECT_SAMPLE_FACTORS           	-	Huh7_elaidic_C18-1trans_3	Sample source:Huh7 cells | Treatment:BSA-elaidate	RAW_FILE_NAME(Raw file name)=Huh7_elaidic_C18-1trans_3.d
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells (~400,000) were spiked with internal standards sphinganine-d7 (Avanti
CO:COLLECTION_SUMMARY            	Polar Lipids, Cat# 860658) and sphingosine-d7 (Avanti Polar Lipids, Cat# 860657)
CO:COLLECTION_SUMMARY            	and were scraped with 0.5 mL methanol.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Huh7 cells were treated with 100 µM BSA-palmitate, BSA-heptadecanoate,
TR:TREATMENT_SUMMARY             	BSA-stearate, BSA-isoC16:0, BSA-anteisoC17:0, BSA-isoC17:0, BSA-oleate, or
TR:TREATMENT_SUMMARY             	BSA-elaidate for 48 hours.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Cells (~400,000) were spiked with internal standards sphinganine-d7 (Avanti
SP:SAMPLEPREP_SUMMARY            	Polar Lipids, Cat# 860658) and sphingosine-d7 (Avanti Polar Lipids, Cat# 860657)
SP:SAMPLEPREP_SUMMARY            	and were scraped with 0.5 mL methanol. Homogenate aliquot of 50 µL was taken to
SP:SAMPLEPREP_SUMMARY            	determine protein content using the BCA protein assay (Thermo Fisher
SP:SAMPLEPREP_SUMMARY            	Scientific). Samples were placed on a mixer for 1 hr at 37°C and then
SP:SAMPLEPREP_SUMMARY            	centrifuged for 5 min at 16,000g. The MeOH was supernatant transferred to a new
SP:SAMPLEPREP_SUMMARY            	Eppendorf tube and hydrolyzed for 16 hr at 65°C. 100 µL 10M KOH, 625 µL
SP:SAMPLEPREP_SUMMARY            	chloroform, 100 µL 2N NH4OH, and 500 µL alkaline water were added to samples
SP:SAMPLEPREP_SUMMARY            	followed by vortexing for 5 min and centrifugation for 5 min at 16,000g. The
SP:SAMPLEPREP_SUMMARY            	lower organic phase was washed 3 times with alkaline water and dried under air.
SP:SAMPLEPREP_SUMMARY            	After dried extracts 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                   	Thermo Hypersil GOLD C18 Selectivity (100 x 2.1 mm, 1.9 µm)
CH:SOLVENT_A                     	60% methanol 40% water; 0.1% formic acid; 5 mM ammonium formate
CH:SOLVENT_B                     	100% methanol; 0.1% formic acid; 5 mM ammonium formate
CH:FLOW_GRADIENT                 	0 min, 40%B; 0.5 min, 40%B; 16 min, 100%B; 25.5 min, 100%B; 26 min, 40%B
CH:FLOW_RATE                     	0.2 ml/min
CH:COLUMN_TEMPERATURE            	40
#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                   	Agilent Masshunter
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Peak area
MS_METABOLITE_DATA_START
Samples	Huh7_palm_C16_1	Huh7_palm_C16_2	Huh7_palm_C16_3	Huh7_C17_1	Huh7_C17_2	Huh7_C17_3	Huh7_stearic_C18_1	Huh7_stearic_C18_2	Huh7_stearic_C18_3	Huh7_isoC16_1	Huh7_isoC16_2	Huh7_isoC16_3	Huh7_anteisoC17_1	Huh7_anteisoC17_2	Huh7_anteisoC17_3	Huh7_isoC17_1	Huh7_isoC17_2	Huh7_isoC17_3	Huh7_oleic_C18-1cis_1	Huh7_oleic_C18-1cis_2	Huh7_oleic_C18-1cis_3	Huh7_elaidic_C18-1trans_1	Huh7_elaidic_C18-1trans_2	Huh7_elaidic_C18-1trans_3			
Factors	Sample source:Huh7 cells | Treatment:BSA-palmitate	Sample source:Huh7 cells | Treatment:BSA-palmitate	Sample source:Huh7 cells | Treatment:BSA-palmitate	Sample source:Huh7 cells | Treatment:BSA-heptadecanoate	Sample source:Huh7 cells | Treatment:BSA-heptadecanoate	Sample source:Huh7 cells | Treatment:BSA-heptadecanoate	Sample source:Huh7 cells | Treatment:BSA-stearate	Sample source:Huh7 cells | Treatment:BSA-stearate	Sample source:Huh7 cells | Treatment:BSA-stearate	Sample source:Huh7 cells | Treatment:BSA-isoC16 0	Sample source:Huh7 cells | Treatment:BSA-isoC16 0	Sample source:Huh7 cells | Treatment:BSA-isoC16 0	Sample source:Huh7 cells | Treatment:BSA-anteisoC17 0	Sample source:Huh7 cells | Treatment:BSA-anteisoC17 0	Sample source:Huh7 cells | Treatment:BSA-anteisoC17 0	Sample source:Huh7 cells | Treatment:BSA-isoC17 0	Sample source:Huh7 cells | Treatment:BSA-isoC17 0	Sample source:Huh7 cells | Treatment:BSA-isoC17 0	Sample source:Huh7 cells | Treatment:BSA-oleate	Sample source:Huh7 cells | Treatment:BSA-oleate	Sample source:Huh7 cells | Treatment:BSA-oleate	Sample source:Huh7 cells | Treatment:BSA-elaidate	Sample source:Huh7 cells | Treatment:BSA-elaidate	Sample source:Huh7 cells | Treatment:BSA-elaidate
SA d18:0 d7	487439	483485	408086	606749	523735	585393	451950	474459	600506	493873	652397	360955	528593	506249	416904	606694	534524	456108	385456	398656	473405	453593	571694	650199			
SA d18:0 iso										1065514	1230201	666064															
SA d18:0	3523535	3286078	2799824	1775676	1669315	1787317	3578812	3120892	3589312	756486	957293	548615	560743	612266	487470	1197030	1257891	1043636	2315259	2648159	2547963	2597841	2625934	2667793			
SA d19:0 anteiso													1708411	1748192	1339358												
SA d19:0 iso																1490964	1488599	1279156									
SA d19:0	28274	23586	18530	1478048	1310276	1465699	47698	39507	47085	8010	11885	5454	13880	22318	12088	30000	25930	7117	32289	39525	38421	33493	35766	35030			
SA d20:0	124933	94718	78199	62755	58033	63841	942317	880434	1111782	24046	38042	17213	55886	41846	26581	46877	46009	33864	77010	95832	91237	103679	98717	123375			
SA d20:1, 11E																						888199	927596	1125764			
SA d20:1, 11Z	59756	44607	32404	10870	7423	28748	74089	58838	80643	3658	20881	1068	4721	8925	1095	16160	15503	7671	78330	89988	101757	20737	19165	26142			
SO d18:1 d7	1641278	1549776	1441947	2024907	1751598	1937900	1491577	1654773	1990372	1664939	1943109	1268755	2255849	2194090	1857100	2020858	1876073	1629124	1293428	1421897	1632613	1564296	1850749	2167655			
SO d18:1 iso										20693034	24461667	13301466															
SO d18:1	49892673	47881829	39846649	31825721	29392406	30805227	44663681	41093452	47952432	22498262	27076184	15341605	27468942	30130068	23587502	28925393	28287398	25299199	33736081	40059893	38055077	35918661	38734855	36756231			
SO d19:1 anteiso													28285330	30759883	24979617												
SO d19:1 iso																24343000	26747539	20695922									
SO d19:1	625766	586907	421489	14850698	12427972	14661117	815954	831394	1011972	205299	262362	138684	258606	172066	228012	1089401	1161857	1074167	739133	939274	1057521	587259	677812	712694			
SO d20:1	2628669	2494825	1876957	1570355	1379482	1584732	13441556	13643549	16524544	651693	921697	435146	994609	962089	684656	1133835	1109721	913708	2798711	3381871	3218098	1944530	2142445	2270642			
SO d20:2, 11E																						5918863	6525083	7069204			
SO d20:2, 11Z	188638	179524	91713	101791	93886	90662	384485	317670	386901	75945	125124	50123	53688	50268	45939	84759	84563	72360	291574	378328	332388	136788	119109	115635			
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Precursor Ion	Product Ion
SA d18:0 d7	309.4	291.5
SA d18:0 iso	302.4	284.4
SA d18:0	302.4	284.4
SA d19:0 anteiso	316.4	298.4
SA d19:0 iso	316.4	298.4
SA d19:0	316.4	298.4
SA d20:0	330.3	312.4
SA d20:1, 11E	328.4	310.4
SA d20:1, 11Z	328.4	310.4
SO d18:1 d7	307.3	289.4
SO d18:1 iso	300.4	282.4
SO d18:1	300.4	282.4
SO d19:1 anteiso	314.4	296.4
SO d19:1 iso	314.4	296.4
SO d19:1	314.4	296.4
SO d20:1	328.4	310.4
SO d20:2, 11E	326.4	308.4
SO d20:2, 11Z	326.4	308.4
METABOLITES_END
#END