#METABOLOMICS WORKBENCH hatalbott2_20191118_161906 DATATRACK_ID:1852 STUDY_ID:ST001286 ANALYSIS_ID:AN002137 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                   	Thermo Hypersil Gold C18 (50 x 1.2mm, 1.7um)
CH:INTERNAL_STANDARD             	C17 Sphingosine, C17 Sphinganine,Cer(d18:1/12:0), GlcCer(d18:1/12:0),
CH:INTERNAL_STANDARD             	LacCer(d18:1/12:0)
#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                      	POSITIVE
MS:MS_COMMENTS                   	sMRM Prec SB frag.
#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
Sphinganine	0	0.074681748	1.109685348
Sphingosine	0	0	0
Cer(d16:1/16:0)	0.26782155	0.148291093	1.024119322
Cer(d16:1/18:0)	0.021927297	0	0.080448647
Cer(d18:1/14:0)	0.650610018	0.363639816	0.819638896
Cer(d18:2/16:0)	0.110654794	0.044851071	1.152945852
Cer(d18:1/16:1)	0	0	0
Cer(d18:1/16:0)	9.071798528	4.592071974	54.02023884
Cer(d18:1/16:0(OH))	2.536959068	0.745097368	1.60959888
Cer(d18:2/18:1)	0.138269196	0.040078403	0.072016877
Cer(d18:1/18:1)	0.529923136	0.390715591	1.301201072
Cer(d18:1/18:0)	0	0	2.501062395
Cer(d18:2/20:1)	0.039979172	0	0.068108833
Cer(d18:2/20:0)	0.065744525	0.004268173	0
Cer(d18:1/20:0)	0	0	0
Cer(d18:2/22:1)	0.129448482	0.019610636	0.051162394
Cer(d18:1/24:1)	14.22200206	8.710743037	77.6661647
Cer(d18:1/24:0)	0	0	44.69866029
Cer(d18:1/25:0)	0	0	0
GlcCer(d18:1/14:0)	0.06555451	0.045076006	0.111433906
Cer(d18:1/26:1)	0	0	0
Cer(d18:1/26:0)	0	0	0
GlcCer(d18:1/16:1)	0	0	0
GlcCer(d18:1/16:0)	1.54852109	1.197628402	6.48766492
GlcCer(d18:1/18:1)	0	0.000516084	0.119684473
GlcCer(d18:1/18:0)	0.429260822	0.126260802	0.900671088
GlcCer(d18:1/20:1)	0	0	0.039419013
GlcCer(d18:1/20:0)	0	0.086107755	0.243094757
GlcCer(d18:1/22:0)	0.627280885	0.744028618	3.518740946
GlcCer(d18:1/24:1)	0.209399747	0.251806011	2.295401581
GlcCer(d18:1/24:0)	0.518468154	0.755765793	3.918735835
GlcCer(d18:1/26:1)	0	0	0
GlcCer(d18:1/26:0)	0	0	0
LacCer(d18:1/16:0)	1.045194528	0.306367937	2.59754704
LacCer(d18:1/18:1)	0.073331926	0.053298592	0
LacCer(d18:1/18:0)	0.097210509	0	0.09068017
LacCer(d18:1/22:0)	0	0	0.338500303
LacCer(d18:1/24:1)	0	0.092433279	0
LacCer(d18:1/24:0)	0	0	0.034537728
LacCer(d18:1/26:1)	0	0	0
LacCer(d18:1/26:0)	0.019520366	0	0.049075767
Cer(d18:0/16:0)	0.827192721	0.959490725	7.166436435
Cer(d18:0/18:1)	0	0	0.040171863
Cer(d18:0/18:0)	0	0	0.195856123
Cer(d18:0/20:0)	0	0	0
Cer(d18:0/22:0)	0	0	0.416537403
Cer(d18:0/24:1)	0	0	1.087529251
Cer(d18:0/24:0)	0	0	0.114948774
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
Sphinganine	C18H39NO2	301.2981	30476		1.8	HMDB0000269	OTKJDMGTUTTYMP-ZWKOTPCHSA-N	LMSP01020001
Sphingosine	C18H37NO2	299.2824	30474		1.52		WWUZIQQURGPMPG-KRWOKUGFSA-N	LMSP01010001
Cer(d16:1/16:0)	C32H63NO3	509.4808			3.97
Cer(d16:1/18:0)	C34H67NO3	537.5121	30603		4.16
Cer(d18:1/14:0)	C32H63NO3	509.4808	30562		3.96
Cer(d18:2/16:0)	C34H65NO3	535.4964	30582		4
Cer(d18:1/16:1)	C34H65NO3	535.4964			4.09
Cer(d18:1/16:0)	C34H67NO3	537.5121	30565		4.16
Cer(d18:1/16:0(OH))	C34H67NO4	553.507			4.09
Cer(d18:2/18:1)	C36H67NO3	561.5121	30583		4.07
Cer(d18:1/18:1)	C36H69NO3	563.5277	30564		4.19
Cer(d18:1/18:0)	C36H71NO3	565.5434	30566		4.33
Cer(d18:2/20:1)	C38H71NO3	589.5434	30585		3.86
Cer(d18:2/20:0)	C38H73NO3	591.559	30584		4.36
Cer(d18:1/20:0)	C38H75NO3	593.5747	30567		4.48
Cer(d18:2/22:1)	C40H75NO3	617.5747			4.39
Cer(d18:1/24:1)	C42H81NO3	647.6216	30569		4.62
Cer(d18:1/24:0)	C42H83NO3	649.6373	30572		4.75
Cer(d18:1/25:0)	C43H85NO3	663.6529	30573		4.84
GlcCer(d18:1/14:0)					3.86
Cer(d18:1/26:1)	C44H85NO3	675.6529	30570		4.78
Cer(d18:1/26:0)	C44H87NO3	677.6686	30571		4.92
GlcCer(d18:1/16:1)	C40H75NO8	697.5493			3.76
GlcCer(d18:1/16:0)	C40H77NO8	699.5649	31063		4.06
GlcCer(d18:1/18:1)	C42H79NO8	725.5806	31080		4.1
GlcCer(d18:1/18:0)	C42H81NO8	727.5962	31065		4.23
GlcCer(d18:1/20:1)	C44H83NO8	753.6119			4.26
GlcCer(d18:1/20:0)	C44H85NO8	755.6275	31066		4.39
GlcCer(d18:1/22:0)	C46H89NO8	783.6588	31067		4.52
GlcCer(d18:1/24:1)	C48H91NO8	809.6745	31068		4.52
GlcCer(d18:1/24:0)	C48H93NO8	811.6901	31069		4.65
GlcCer(d18:1/26:1)	C50H95NO8	837.7058	31070		4.66
GlcCer(d18:1/26:0)	C50H97NO8	839.7214	31071		4.8
LacCer(d18:1/16:0)	C46H87NO13	861.6177	31136		4
LacCer(d18:1/18:1)	C48H89NO13	887.6334	31144		4.06
LacCer(d18:1/18:0)	C48H91NO13	889.649	31137		4.19
LacCer(d18:1/22:0)	C52H99NO13	945.7116	31139		4.49
LacCer(d18:1/24:1)	C54H101NO13	971.7273	31142		4.49
LacCer(d18:1/24:0)	C54H103NO13	973.7429	31140		4.59
LacCer(d18:1/26:1)	C56H105NO13	999.7586	31143		4.63
LacCer(d18:1/26:0)	C56H107NO13	1001.7742	31141		4.76
Cer(d18:0/16:0)	C34H69NO3	539.5277	30653		4.21
Cer(d18:0/18:1)	C36H71NO3	565.5434	30661		4.26
Cer(d18:0/18:0)	C36H73NO3	567.559	30654		4.37
Cer(d18:0/20:0)	C38H77NO3	595.5903	30655		4.46
Cer(d18:0/22:0)	C40H81NO3	623.6216	30656		4.59
Cer(d18:0/24:1)	C42H83NO3	649.6373	30657		4.65
Cer(d18:0/24:0)	C42H85NO3	651.6529	30658		4.78
METABOLITES_END
#END