#METABOLOMICS WORKBENCH hatalbott2_20191118_161906 DATATRACK_ID:1852 STUDY_ID:ST001286 ANALYSIS_ID:AN002136 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                      	POSITIVE
MS:MS_COMMENTS                   	sMRM Prec 184 u (KOH)
#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
SM(d18:1/14:0)	5.525509227	3.572569737	31.13467598
SM(d18:0/14:0)	0.096740025	0.134956575	1.139515909
SM(d18:1/16:1)	5.50684522	4.069120915	27.24593189
SM(d18:1/16:0)	137.87794	102.0558851	796.0217512
SM(d18:0/16:0)	4.807548003	2.641567635	18.59713689
SM(d18:0/18:1)	0.211888623	0.058206595	0.649904544
SM(d18:1/18:1)	3.011813681	1.857571491	12.05078486
SM(d18:1/18:0)	18.31518505	12.29159273	80.8356873
SM(d18:0/18:0)	0.489836094	0.173248059	3.71365408
SM(d18:1/20:0)	0	0	0
SM(d18:0/20:0)	0	0	0
SM(d18:1/22:1)	3.396590599	1.920461054	18.82563873
SM(d18:1/23:0)	22.00908718	17.38795819	126.5314508
SM(d18:1/24:2)	5.619704837	4.287478522	30.05939041
SM(d18:1/24:1)	20.77026939	14.67842087	112.752746
SM(d18:1/24:0)	20.23669541	16.2351697	110.2439133
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
SM(d18:1/14:0)	C37H75N2O6P	674.5363	30744		3.02
SM(d18:0/14:0)	C37H77N2O6P	676.5519	30746		3.13
SM(d18:1/16:1)	C39H77N2O6P	700.5519	30755		3.09
SM(d18:1/16:0)	C39H79N2O6P	702.5676	30728		3.26
SM(d18:0/16:0)	C39H81N2O6P	704.5832	30729		3.35
SM(d18:0/18:1)	C41H83N2O6P	730.5989	30747		3.32
SM(d18:1/18:1)	C41H81N2O6P	728.5832	30745		3.17
SM(d18:1/18:0)	C41H83N2O6P	730.5989	30726		3.47
SM(d18:0/18:0)	C41H85N2O6P	732.6145	30736		3.54
SM(d18:1/20:0)	C43H87N2O6P	758.6302	30730		3.4
SM(d18:0/20:0)	C43H89N2O6P	760.6458	30737		3.5
SM(d18:1/22:1)	C45H89N2O6P	784.6458	30785		3.71
SM(d18:1/23:0)	C46H93N2O6P	800.6771	30791		3.78
SM(d18:1/24:2)	C47H91N2O6P	810.6615			3.74
SM(d18:1/24:1)	C47H93N2O6P	812.6771	30732		3.9
SM(d18:1/24:0)	C47H95N2O6P	814.6928	30733		4.07
METABOLITES_END
#END