#METABOLOMICS WORKBENCH hatalbott2_20191118_161906 DATATRACK_ID:1852 STUDY_ID:ST001286 ANALYSIS_ID:AN002135 PROJECT_ID:PR000868
VERSION             	1
CREATED_ON             	December 12, 2019, 5:08 pm
#PROJECT
PR:PROJECT_TITLE                 	Lipid composition of isolated lipid droplets from the functional bovine corpus
PR:PROJECT_TITLE                 	luteum
PR:PROJECT_TYPE                  	lipidomics
PR:PROJECT_SUMMARY               	Establishment and maintenance of pregnancy is dependent on progesterone
PR:PROJECT_SUMMARY               	synthesized by the corpus luteum (CL). The CL is known for the prominent
PR:PROJECT_SUMMARY               	presence of intracellular lipid droplets (LDs). However relatively little is
PR:PROJECT_SUMMARY               	known about the composition and function of these luteal LDs. Our objective was
PR:PROJECT_SUMMARY               	to identify the lipid composition of LDs from fully functional bovine CLs.
PR:PROJECT_SUMMARY               	Luteal LDs were isolated by flotation through a discontinuous sucrose gradient,
PR:PROJECT_SUMMARY               	lipids were then extracted using a standard Bligh and Dyer protocol, dried, and
PR:PROJECT_SUMMARY               	sent to Avanti Polar Lipids for lipidomics analysis. The samples were provided
PR:PROJECT_SUMMARY               	for lipidomic profiling of free sterols, cholesteryl esters, triglycerides,
PR:PROJECT_SUMMARY               	diacylglycerols, phospholipids, and sphingolipids. Molecular species were
PR:PROJECT_SUMMARY               	resolved by reversed-phase liquid chromatography in the presence of class and
PR:PROJECT_SUMMARY               	sub-class specific internal standard compounds added to each sample. The
PR:PROJECT_SUMMARY               	compounds were detected by tandem mass spectrometry (MS/MS) with scheduled
PR:PROJECT_SUMMARY               	multiple reaction monitoring (MRM) for mass-specific fragment ions according to
PR:PROJECT_SUMMARY               	the lipid class and molecular weight of the compound. Quantification of
PR:PROJECT_SUMMARY               	cholesterol, cholesteryl esters, triglycerides, and diglycerides were directly
PR:PROJECT_SUMMARY               	calculated with standards and internal standards from calibration response
PR:PROJECT_SUMMARY               	curves. The remaining lipid species were semi-quantization using the integrated
PR:PROJECT_SUMMARY               	area of each analyte’s MRM peak, divided by the appropriate internal standard
PR:PROJECT_SUMMARY               	peak area, and multiplied by the standard’s known concentration. Lipid
PR:PROJECT_SUMMARY               	concentrations were normalized to the corresponding protein concentration of
PR:PROJECT_SUMMARY               	each sample and as a mol % relative to total lipids or within each lipid class.
PR:PROJECT_SUMMARY               	Isolated luteal LDs were composed primarily of triglyceride (88%, mol% of lipid
PR:PROJECT_SUMMARY               	class to total lipids). Other neutral lipids included diacylglycerol, 2.9%; and
PR:PROJECT_SUMMARY               	cholesteryl esters, 1.5%. Polar lipids were primarily composed of
PR:PROJECT_SUMMARY               	phosphatidylcholine (3.1%), sphingomyelin (1.5%), phosphatidylinositol (0.9%),
PR:PROJECT_SUMMARY               	phosphatidylethanolamine (0.8%) and phosphatidylserine (0.4%). A number of other
PR:PROJECT_SUMMARY               	minor lipids representing less than 0.32% of the total lipid pool were also
PR:PROJECT_SUMMARY               	detected including phosphatidylglycerol, lysophospholipids, ceramides, and
PR:PROJECT_SUMMARY               	glycosylated ceramides. Lipid composition of bovine luteal LDs are distinct from
PR:PROJECT_SUMMARY               	LDs isolated from other tissues and in other species.
PR:INSTITUTE                     	University of Nebraska Medical Center
PR:DEPARTMENT                    	Obstetrics and Gynecology
PR:LABORATORY                    	John S. Davis
PR:LAST_NAME                     	Davis
PR:FIRST_NAME                    	John
PR:ADDRESS                       	983255 Nebraska Medical Center Omaha, NE 68198-3255
PR:EMAIL                         	jsdavis@unmc.edu
PR:PHONE                         	402-599-9079
PR:FUNDING_SOURCE                	INBRE - P20GM103427-14, COBRE - 1P30GM110768-01
PR:CONTRIBUTORS                  	Heather Talbott, Xiaoying Hou, Crystal Cordes
#STUDY
ST:STUDY_TITLE                   	Lipid composition of isolated lipid droplets from the functional bovine corpus
ST:STUDY_TITLE                   	luteum
ST:STUDY_TYPE                    	Lipidomics
ST:STUDY_SUMMARY                 	Establishment and maintenance of pregnancy is dependent on progesterone
ST:STUDY_SUMMARY                 	synthesized by the corpus luteum (CL). The CL is known for the prominent
ST:STUDY_SUMMARY                 	presence of intracellular lipid droplets (LDs). However relatively little is
ST:STUDY_SUMMARY                 	known about the composition and function of these luteal LDs. Our objective was
ST:STUDY_SUMMARY                 	to identify the lipid composition of LDs from fully functional bovine CLs.
ST:STUDY_SUMMARY                 	Luteal LDs were isolated by flotation through a discontinuous sucrose gradient,
ST:STUDY_SUMMARY                 	lipids were then extracted using a standard Bligh and Dyer protocol, dried, and
ST:STUDY_SUMMARY                 	sent to Avanti Polar Lipids for lipidomics analysis. The samples were provided
ST:STUDY_SUMMARY                 	for lipidomic profiling of free sterols, cholesteryl esters, triglycerides,
ST:STUDY_SUMMARY                 	diacylglycerols, phospholipids, and sphingolipids. Molecular species were
ST:STUDY_SUMMARY                 	resolved by reversed-phase liquid chromatography in the presence of class and
ST:STUDY_SUMMARY                 	sub-class specific internal standard compounds added to each sample. The
ST:STUDY_SUMMARY                 	compounds were detected by tandem mass spectrometry (MS/MS) with scheduled
ST:STUDY_SUMMARY                 	multiple reaction monitoring (MRM) for mass-specific fragment ions according to
ST:STUDY_SUMMARY                 	the lipid class and molecular weight of the compound. Quantification of
ST:STUDY_SUMMARY                 	cholesterol, cholesteryl esters, triglycerides, and diglycerides were directly
ST:STUDY_SUMMARY                 	calculated with standards and internal standards from calibration response
ST:STUDY_SUMMARY                 	curves. The remaining lipid species were semi-quantization using the integrated
ST:STUDY_SUMMARY                 	area of each analyte’s MRM peak, divided by the appropriate internal standard
ST:STUDY_SUMMARY                 	peak area, and multiplied by the standard’s known concentration. Lipid
ST:STUDY_SUMMARY                 	concentrations were normalized to the corresponding protein concentration of
ST:STUDY_SUMMARY                 	each sample and as a mol % relative to total lipids or within each lipid class.
ST:STUDY_SUMMARY                 	Isolated luteal LDs were composed primarily of triglyceride (88%, mol% of lipid
ST:STUDY_SUMMARY                 	class to total lipids). Other neutral lipids included diacylglycerol, 2.9%; and
ST:STUDY_SUMMARY                 	cholesteryl esters, 1.5%. Polar lipids were primarily composed of
ST:STUDY_SUMMARY                 	phosphatidylcholine (3.1%), sphingomyelin (1.5%), phosphatidylinositol (0.9%),
ST:STUDY_SUMMARY                 	phosphatidylethanolamine (0.8%) and phosphatidylserine (0.4%). A number of other
ST:STUDY_SUMMARY                 	minor lipids representing less than 0.32% of the total lipid pool were also
ST:STUDY_SUMMARY                 	detected including phosphatidylglycerol, lysophospholipids, ceramides, and
ST:STUDY_SUMMARY                 	glycosylated ceramides. Lipid composition of bovine luteal LDs are distinct from
ST:STUDY_SUMMARY                 	LDs isolated from other tissues and in other species.
ST:INSTITUTE                     	University of Nebraska Medical Center
ST:DEPARTMENT                    	Obstetrics and Gynecology
ST:LABORATORY                    	John S. Davis
ST:LAST_NAME                     	Davis
ST:FIRST_NAME                    	John
ST:ADDRESS                       	983255 Nebraska Medical Center Omaha, NE 68198-3255
ST:EMAIL                         	jsdavis@unmc.edu
ST:PHONE                         	402-559-9079
ST:NUM_GROUPS                    	1
ST:TOTAL_SUBJECTS                	3
ST:NUM_FEMALES                   	3
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Bos taurus
SU:TAXONOMY_ID                   	9913
SU:GENDER                        	Female
SU:ANIMAL_ANIMAL_SUPPLIER        	JBS Beef Plant 3435 Edward Babe Gomez Ave, Omaha, NE 68107
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	bovine_CL_LD_replicate1	Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	bovine_CL_LD_replicate2	Treatment:Control	
SUBJECT_SAMPLE_FACTORS           	-	bovine_CL_LD_replicate3	Treatment:Control	
#COLLECTION
CO:COLLECTION_SUMMARY            	Tissue (~2.5 g) was washed thoroughly in TE buffer (10 mM Tris, 1 mM EDTA, pH
CO:COLLECTION_SUMMARY            	7.4). Minced tissue was resuspended in 10 mL tissue homogenate buffer (60%
CO:COLLECTION_SUMMARY            	sucrose w/v in TE buffer containing protease and phosphatase inhibitor
CO:COLLECTION_SUMMARY            	cocktails) and homogenized with a Teflon Dounce homogenizer in a glass vessel.
CO:COLLECTION_SUMMARY            	The post-nuclear supernatant (PNS) fraction was obtained after centrifugation at
CO:COLLECTION_SUMMARY            	2000 rcf for 10 min. The supernatant was loaded into a 30 mL ultracentrifuge
CO:COLLECTION_SUMMARY            	tube and overlaid sequentially with 40%, 25%, 10%, and 0% sucrose w/v in TE
CO:COLLECTION_SUMMARY            	buffer containing protease and phosphatase inhibitor cocktails. Samples were
CO:COLLECTION_SUMMARY            	centrifuged at 110,000 × g (ravg) for 30 min at 4 °C with no brake in a
CO:COLLECTION_SUMMARY            	Beckman Coulter Avanti J-20 XP ultracentrifuge using an SW 32 Ti rotor. The LDs
CO:COLLECTION_SUMMARY            	concentrated in a yellow-ish band at the top of the gradient were harvested and
CO:COLLECTION_SUMMARY            	concentrated by centrifugation at 2000 rcf for 10 min at 4 °C. This protocol
CO:COLLECTION_SUMMARY            	was derived from Ding et al. 2012, and Brasaemale et al. 2016. Ding, Y., Zhang,
CO:COLLECTION_SUMMARY            	S., Yang, L., Na, H., Zhang, P., Zhang, H., … Liu, P. (2013). Isolating lipid
CO:COLLECTION_SUMMARY            	droplets from multiple species. Nature Protocols, 8(1), 43–51.
CO:COLLECTION_SUMMARY            	https://doi.org/10.1038/nprot.2012.142 Brasaemle, D. L., & Wolins, N. E. (2016).
CO:COLLECTION_SUMMARY            	Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation.
CO:COLLECTION_SUMMARY            	Current Protocols in Cell Biology, 72, 3.15.1-3.15.13.
CO:COLLECTION_SUMMARY            	https://doi.org/10.1002/cpcb.10
CO:SAMPLE_TYPE                   	Ovary
CO:VOLUMEORAMOUNT_COLLECTED      	2.5 g of corpus luteum tissue
#TREATMENT
TR:TREATMENT_SUMMARY             	N/A
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Lipids from CL tissue LDs (~250uL) were extracted using a standard Bligh and
SP:SAMPLEPREP_SUMMARY            	Dyer extraction protocol and then dried and sent to Avanti Polar Lipids for
SP:SAMPLEPREP_SUMMARY            	lipidomics analysis. Extracts were received as dried residues in glass vials and
SP:SAMPLEPREP_SUMMARY            	were immediately stored at -80 °C until analysis. Bligh, E. G., & Dyer, W. J.
SP:SAMPLEPREP_SUMMARY            	(1959). A rapid method of total lipid extraction and purification. Canadian
SP:SAMPLEPREP_SUMMARY            	Journal of Biochemistry and Physiology, 37(8), 911–917.
SP:SAMPLEPREP_SUMMARY            	https://doi.org/10.1139/o59-099
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACTION_METHOD             	Bligh & Dyer, chloroform:methanol (1:2, v:v)
SP:EXTRACT_STORAGE               	-80℃
SP:SAMPLE_RESUSPENSION           	1mL of chloroform:methanol (8:2, v/v)
SP:SAMPLE_DERIVATIZATION         	N/A
SP:SUBCELLULAR_LOCATION          	Lipid Droplet
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Molecular species were resolved by reversed-phase liquid chromatography in the
CH:CHROMATOGRAPHY_SUMMARY        	presence of class and sub-class specific internal standard compounds added to
CH:CHROMATOGRAPHY_SUMMARY        	each sample. Selectivity was further enhanced by scheduling the detection of
CH:CHROMATOGRAPHY_SUMMARY        	each compound according to its elution from the high-performance liquid
CH:CHROMATOGRAPHY_SUMMARY        	chromatography (HPLC) column, known as scheduled MRM (sMRM).
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Agilent Eclipse XBD C8 (50 x 4.6mm, 1.8um)
CH:INTERNAL_STANDARD             	LPC(17:0), PC(37:4), LPE(17:1), PE(37:4)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	Avanti Polar Lipids, Inc
AN:DETECTOR_TYPE                 	AcQuRate™ Pulse Counting CEM
#MS
MS:INSTRUMENT_NAME               	ABI Sciex 5500 QTrap
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	sMRM NL 87 u
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	nM
MS_METABOLITE_DATA_START
Samples	bovine_CL_LD_replicate1	bovine_CL_LD_replicate2	bovine_CL_LD_replicate3
Factors	Treatment:Control	Treatment:Control	Treatment:Control
LPS(16:0)	0.108918988	0	0
LPS(18:3)	0.02299992	0	0
LPS(18:2)	0.046906267	0	0.023497528
LPS(18:1)	0.348490907	0.24972622	0.761588497
LPS(18:0)	0.412735164	0.313883621	1.249784589
LPS(20:4)	0	0	0
LPS(20:3)	0	0.058191563	0.137916738
LPS(20:2)	0.001386176	0	0.013713266
LPS(20:1)	0.001505687	0.006136273	0
LPS(20:0)	0	0	0
LPS(22:6)	0.386530906	0.253588118	0.989022756
PS(34:2)	0.01746611	0.33424515	1.437490869
PS(34:1)	1.274937901	1.264905801	6.691799338
PS(34:0)	0.182407093	0	0.830697061
PS(36:4)	0	0	1.079693882
PS(36:3)	1.00795523	0.588666739	3.713239524
PS(36:2)	4.732651206	3.617113571	19.11724807
PS(36:1)	7.516913985	9.511472884	52.19091413
PS(36:0)	0.033665114	0	0.075882904
PS(38:6)	0	0	0.098108747
PS(38:5)	0.872219971	0.663131335	4.36663133
PS(38:4)	2.385256595	2.020705667	12.16601613
PS(38:3)	5.164240615	8.78451058	36.65639315
PS(38:2)	1.498481612	2.605030533	16.58765906
PS(38:1)	0.538753509	0.964504941	4.296959154
PS(40:7)	0	0	0
PS(40:6)	0.237663282	0.613911417	5.979563369
PS(40:5)	16.43385681	16.21417012	101.5532788
PS(40:4)	5.685870431	13.28870945	50.83893961
PS(40:3)	0.027536724	0	3.046742588
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Formula	Mass	MW structure	Mass Info (precursor ion, product ion)	retention times	Human Metabolome Database	InChIKey	LipidMAPS	quantified m/z
LPS(16:0)	C22H42NO10P	511.25		496.3 / 409.3	1.04		XIVOBOJQPNEUSC-UXHICEINSA-N	LMGP03050002	496.3
LPS(18:3)				518.3 / 431.3	0.77				518.3
LPS(18:2)	C24H42NO10P	535.25		520.3 / 433.3	0.99				520.3
LPS(18:1)	C24H44NO10P	537.27		522.3 / 435.3	1.22		JZWNYZVVZXZRRH-YFKVPUFHSA-N	LMGP03050001	522.3
LPS(18:0)	C24H46NO10P	539.29		524.3 / 437.3	1.32		ZPDQFUYPBVXUKS-YADHBBJMSA-N	LMGP03050006	524.3
LPS(20:4)	C26H42NO10P	559.25		544.3 / 457.3	0.99		XHWSRRGLFMDBOB-RRJHOXOUSA-N	LMGP03050007	544.3
LPS(20:3)	C26H44NO10P	561.27		546.3 / 459.3	0.95				546.3
LPS(20:2)	C26H46NO10P	563.29		548.3 / 461.3	1.54				548.3
LPS(20:1)	C26H48NO10P	565.3		550.3 / 463.3	2.33				550.3
LPS(20:0)	C26H50NO10P	567.32		552.3 / 465.3	2.75				552.3
LPS(22:6)				570.3 / 483.3	0.99				570.3
PS(34:2)	C40H74NO10P	759.51		758.5 / 671.5	3.06	HMDB0012388	JSCZUPSIMWRJHP-KQQJSZDRSA-N	LMGP03010881	758.5
PS(34:1)	C40H76NO10P	761.52		760.5 / 673.5	3.16	HMDB0012387	ILJAXXNZNFOOQA-DAQGAKHBSA-N	LMGP03010959	760.5
PS(34:0)	C40H78NO10P	763.54		762.5 / 675.5	3.63		UYGORIHCWHGAJE-AARKOHAPSA-N	LMGP03010906	762.5
PS(36:4)	C42H74NO10P	783.51		782.5 / 695.5	3.19	HMDB0012402			782.5
PS(36:3)	C42H76NO10P	785.52		784.5 / 697.5	3.3	HMDB0012391	MWONMGIZXLAUBR-QUBHBNJHSA-N	LMGP03010958	784.5
PS(36:2)	C42H78NO10P	787.54		786.5 / 699.5	3.52	HMDB0012390	WTBFLCSPLLEDEM-JIDRGYQWSA-N	LMGP03010030	786.5
PS(36:1)	C42H80NO10P	789.55		788.5 / 701.5	3.63	HMDB0010163	AJFWREUFUPEYII-PAHWMLEVSA-N	LMGP03010025	788.5
PS(36:0)	C42H82NO10P	791.57		790.5 / 703.5	3.42	HMDB0012378	TZCPCKNHXULUIY-RGULYWFUSA-N	LMGP03010036	790.5
PS(38:6)	C44H74NO10P	807.51		806.5 / 719.5	3.05	HMDB0012362	PWBBJQOVCTWPIM-FAYDGCQZSA-N	LMGP03010043	806.5
PS(38:5)	C44H76NO10P	809.52		808.5 / 721.5	3.07	HMDB0012394	PVENTVZLUAWWEI-OXBTWZGPSA-N	LMGP03010879	808.5
PS(38:4)	C44H78NO10P	811.54		810.5 / 723.5	3.52	HMDB0012383	SVOUGFFDROZBJI-DNALCEECSA-N	LMGP03010039	810.5
PS(38:3)	C44H80NO10P	813.55		812.5 / 725.5	3.33	HMDB0012382	LIBQKAKFGGIIDU-NVSSCHKGSA-N		812.5
PS(38:2)	C44H82NO10P	815.57		814.5 / 727.5	3.74				814.5
PS(38:1)	C44H84NO10P	817.58		816.5 / 729.5	3.56				816.5
PS(40:7)	C46H76NO10P	833.52		832.5 / 745.5	3.3	HMDB0012437	IEHVRLNIHFLLMS-WUDDRQCASA-N	LMGP03010641	832.5
PS(40:6)	C46H78NO10P	835.54		834.5 / 747.5	3.41	HMDB0010167	LYYHRRPTEXPVOR-SYEOQEKUSA-N	LMGP03010040	834.5
PS(40:5)	C46H80NO10P	837.55		836.5 / 749.5	3.32	HMDB0010166	PZEFRSYCROBXSJ-AOWFZNJASA-N		836.5
PS(40:4)	C46H82NO10P	839.57		838.5 / 751.5	3.63				838.5
PS(40:3)	C46H84NO10P	841.58		840.5 / 753.5	3.85				840.5
METABOLITES_END
#END