Summary of study ST001286

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000868. The data can be accessed directly via it's Project DOI: 10.21228/M8GT28 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001286
Study TitleLipid composition of isolated lipid droplets from the functional bovine corpus luteum
Study TypeLipidomics
Study SummaryEstablishment and maintenance of pregnancy is dependent on progesterone synthesized by the corpus luteum (CL). The CL is known for the prominent presence of intracellular lipid droplets (LDs). However relatively little is known about the composition and function of these luteal LDs. Our objective was to identify the lipid composition of LDs from fully functional bovine CLs. Luteal LDs were isolated by flotation through a discontinuous sucrose gradient, lipids were then extracted using a standard Bligh and Dyer protocol, dried, and sent to Avanti Polar Lipids for lipidomics analysis. The samples were provided for lipidomic profiling of free sterols, cholesteryl esters, triglycerides, diacylglycerols, phospholipids, and sphingolipids. Molecular species were resolved by reversed-phase liquid chromatography in the presence of class and sub-class specific internal standard compounds added to each sample. The compounds were detected by tandem mass spectrometry (MS/MS) with scheduled multiple reaction monitoring (MRM) for mass-specific fragment ions according to the lipid class and molecular weight of the compound. Quantification of cholesterol, cholesteryl esters, triglycerides, and diglycerides were directly calculated with standards and internal standards from calibration response curves. The remaining lipid species were semi-quantization using the integrated area of each analyte’s MRM peak, divided by the appropriate internal standard peak area, and multiplied by the standard’s known concentration. Lipid concentrations were normalized to the corresponding protein concentration of each sample and as a mol % relative to total lipids or within each lipid class. Isolated luteal LDs were composed primarily of triglyceride (88%, mol% of lipid class to total lipids). Other neutral lipids included diacylglycerol, 2.9%; and cholesteryl esters, 1.5%. Polar lipids were primarily composed of phosphatidylcholine (3.1%), sphingomyelin (1.5%), phosphatidylinositol (0.9%), phosphatidylethanolamine (0.8%) and phosphatidylserine (0.4%). A number of other minor lipids representing less than 0.32% of the total lipid pool were also detected including phosphatidylglycerol, lysophospholipids, ceramides, and glycosylated ceramides. Lipid composition of bovine luteal LDs are distinct from LDs isolated from other tissues and in other species.
Institute
University of Nebraska Medical Center
DepartmentObstetrics and Gynecology
LaboratoryJohn S. Davis
Last NameDavis
First NameJohn
Address983255 Nebraska Medical Center Omaha, NE 68198-3255
Emailjsdavis@unmc.edu
Phone402-559-9079
Submit Date2019-11-18
Num Groups1
Total Subjects3
Num Females3
Analysis Type DetailLC-MS
Release Date2020-05-18
Release Version1
John Davis John Davis
https://dx.doi.org/10.21228/M8GT28
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000868
Project DOI:doi: 10.21228/M8GT28
Project Title:Lipid composition of isolated lipid droplets from the functional bovine corpus luteum
Project Type:lipidomics
Project Summary:Establishment and maintenance of pregnancy is dependent on progesterone synthesized by the corpus luteum (CL). The CL is known for the prominent presence of intracellular lipid droplets (LDs). However relatively little is known about the composition and function of these luteal LDs. Our objective was to identify the lipid composition of LDs from fully functional bovine CLs. Luteal LDs were isolated by flotation through a discontinuous sucrose gradient, lipids were then extracted using a standard Bligh and Dyer protocol, dried, and sent to Avanti Polar Lipids for lipidomics analysis. The samples were provided for lipidomic profiling of free sterols, cholesteryl esters, triglycerides, diacylglycerols, phospholipids, and sphingolipids. Molecular species were resolved by reversed-phase liquid chromatography in the presence of class and sub-class specific internal standard compounds added to each sample. The compounds were detected by tandem mass spectrometry (MS/MS) with scheduled multiple reaction monitoring (MRM) for mass-specific fragment ions according to the lipid class and molecular weight of the compound. Quantification of cholesterol, cholesteryl esters, triglycerides, and diglycerides were directly calculated with standards and internal standards from calibration response curves. The remaining lipid species were semi-quantization using the integrated area of each analyte’s MRM peak, divided by the appropriate internal standard peak area, and multiplied by the standard’s known concentration. Lipid concentrations were normalized to the corresponding protein concentration of each sample and as a mol % relative to total lipids or within each lipid class. Isolated luteal LDs were composed primarily of triglyceride (88%, mol% of lipid class to total lipids). Other neutral lipids included diacylglycerol, 2.9%; and cholesteryl esters, 1.5%. Polar lipids were primarily composed of phosphatidylcholine (3.1%), sphingomyelin (1.5%), phosphatidylinositol (0.9%), phosphatidylethanolamine (0.8%) and phosphatidylserine (0.4%). A number of other minor lipids representing less than 0.32% of the total lipid pool were also detected including phosphatidylglycerol, lysophospholipids, ceramides, and glycosylated ceramides. Lipid composition of bovine luteal LDs are distinct from LDs isolated from other tissues and in other species.
Institute:University of Nebraska Medical Center
Department:Obstetrics and Gynecology
Laboratory:John S. Davis
Last Name:Davis
First Name:John
Address:983255 Nebraska Medical Center Omaha, NE 68198-3255
Email:jsdavis@unmc.edu
Phone:402-599-9079
Funding Source:INBRE - P20GM103427-14, COBRE - 1P30GM110768-01
Contributors:Heather Talbott, Xiaoying Hou, Crystal Cordes

Subject:

Subject ID:SU001358
Subject Type:Mammal
Subject Species:Bos taurus
Taxonomy ID:9913
Gender:Female
Animal Animal Supplier:JBS Beef Plant 3435 Edward Babe Gomez Ave, Omaha, NE 68107

Factors:

Subject type: Mammal; Subject species: Bos taurus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA093605bovine_CL_LD_replicate3Control
SA093606bovine_CL_LD_replicate2Control
SA093607bovine_CL_LD_replicate1Control
Showing results 1 to 3 of 3

Collection:

Collection ID:CO001352
Collection Summary:Tissue (~2.5 g) was washed thoroughly in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4). Minced tissue was resuspended in 10 mL tissue homogenate buffer (60% sucrose w/v in TE buffer containing protease and phosphatase inhibitor cocktails) and homogenized with a Teflon Dounce homogenizer in a glass vessel. The post-nuclear supernatant (PNS) fraction was obtained after centrifugation at 2000 rcf for 10 min. The supernatant was loaded into a 30 mL ultracentrifuge tube and overlaid sequentially with 40%, 25%, 10%, and 0% sucrose w/v in TE buffer containing protease and phosphatase inhibitor cocktails. Samples were centrifuged at 110,000 × g (ravg) for 30 min at 4 °C with no brake in a Beckman Coulter Avanti J-20 XP ultracentrifuge using an SW 32 Ti rotor. The LDs concentrated in a yellow-ish band at the top of the gradient were harvested and concentrated by centrifugation at 2000 rcf for 10 min at 4 °C. This protocol was derived from Ding et al. 2012, and Brasaemale et al. 2016. Ding, Y., Zhang, S., Yang, L., Na, H., Zhang, P., Zhang, H., … Liu, P. (2013). Isolating lipid droplets from multiple species. Nature Protocols, 8(1), 43–51. https://doi.org/10.1038/nprot.2012.142 Brasaemle, D. L., & Wolins, N. E. (2016). Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation. Current Protocols in Cell Biology, 72, 3.15.1-3.15.13. https://doi.org/10.1002/cpcb.10
Sample Type:Ovary
Volumeoramount Collected:2.5 g of corpus luteum tissue

Treatment:

Treatment ID:TR001373
Treatment Summary:N/A

Sample Preparation:

Sampleprep ID:SP001366
Sampleprep Summary:Lipids from CL tissue LDs (~250uL) were extracted using a standard Bligh and Dyer extraction protocol and then dried and sent to Avanti Polar Lipids for lipidomics analysis. Extracts were received as dried residues in glass vials and were immediately stored at -80 °C until analysis. Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37(8), 911–917. https://doi.org/10.1139/o59-099
Processing Storage Conditions:-80℃
Extraction Method:Bligh & Dyer, chloroform:methanol (1:2, v:v)
Extract Storage:-80℃
Sample Resuspension:1mL of chloroform:methanol (8:2, v/v)
Sample Derivatization:N/A
Subcellular Location:Lipid Droplet

Combined analysis:

Analysis ID AN002130 AN002131 AN002132 AN002133 AN002134 AN002135 AN002136 AN002137 AN002138
Analysis type MS MS MS MS MS MS MS MS MS
Chromatography type Reversed phase Reversed phase Reversed phase Reversed phase Reversed phase Reversed phase Reversed phase Reversed phase Reversed phase
Chromatography system Waters Acquity Waters Acquity Waters Acquity Waters Acquity Waters Acquity Waters Acquity Waters Acquity Waters Acquity Waters Acquity
Column Waters Acquity BEH C18 (50 x 1.2mm, 1.7um) Agilent Eclipse XBD C8 (50 x 4.6mm, 1.8um) Agilent Eclipse XBD C8 (50 x 4.6mm, 1.8um) Agilent Eclipse XBD C8 (50 x 4.6mm, 1.8um) Agilent Eclipse XBD C8 (50 x 4.6mm, 1.8um) Agilent Eclipse XBD C8 (50 x 4.6mm, 1.8um) Agilent Eclipse XBD C8 (50 x 4.6mm, 1.8um) Thermo Hypersil Gold C18 (50 x 1.2mm, 1.7um) Agilent Eclipse XDB-PLUS C18 (50 x 1.2mm, 1.8um)
MS Type ESI ESI ESI ESI ESI ESI ESI ESI ESI
MS instrument type Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole
MS instrument name ABI Sciex 5500 QTrap ABI Sciex 5500 QTrap ABI Sciex 5500 QTrap ABI Sciex 5500 QTrap ABI Sciex 5500 QTrap ABI Sciex 5500 QTrap ABI Sciex 5500 QTrap ABI Sciex 5500 QTrap ABI Sciex 5500 QTrap
Ion Mode POSITIVE POSITIVE POSITIVE NEGATIVE POSITIVE NEGATIVE POSITIVE POSITIVE POSITIVE
Units nM nM nM nM nM nM nM nM nM

Chromatography:

Chromatography ID:CH001562
Chromatography Summary:Molecular species were resolved by reversed-phase liquid chromatography in the presence of class and sub-class specific internal standard compounds added to each sample. Selectivity was further enhanced by scheduling the detection of each compound according to its elution from the high-performance liquid chromatography (HPLC) column, known as scheduled MRM (sMRM).
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (50 x 1.2mm, 1.7um)
Internal Standard:DG(28:0)-d5, DG(30:0)-d5, DG(32:0)-d5, DG(34:0)-d5, DG(38:0)-d5, DG(40:10)-d5, DG(40:8)-d5, DG(40:4)-d5, DG(40:0)-d5, TG(44:1)-d5, TG(48:1)-d5, TG(50:0)-d5, TG(51:1)-d5, TG(58:10)-d5, TG(58:7)-d5, TG(62:16)-d5, TG(60:1)-d5
Chromatography Type:Reversed phase
  
Chromatography ID:CH001563
Chromatography Summary:Molecular species were resolved by reversed-phase liquid chromatography in the presence of class and sub-class specific internal standard compounds added to each sample. Selectivity was further enhanced by scheduling the detection of each compound according to its elution from the high-performance liquid chromatography (HPLC) column, known as scheduled MRM (sMRM).
Instrument Name:Waters Acquity
Column Name:Agilent Eclipse XBD C8 (50 x 4.6mm, 1.8um)
Internal Standard:LPC(17:0), PC(37:4), LPE(17:1), PE(37:4)
Chromatography Type:Reversed phase
  
Chromatography ID:CH001564
Chromatography Summary:Molecular species were resolved by reversed-phase liquid chromatography in the presence of class and sub-class specific internal standard compounds added to each sample. Selectivity was further enhanced by scheduling the detection of each compound according to its elution from the high-performance liquid chromatography (HPLC) column, known as scheduled MRM (sMRM).
Instrument Name:Waters Acquity
Column Name:Thermo Hypersil Gold C18 (50 x 1.2mm, 1.7um)
Internal Standard:C17 Sphingosine, C17 Sphinganine,Cer(d18:1/12:0), GlcCer(d18:1/12:0), LacCer(d18:1/12:0)
Chromatography Type:Reversed phase
  
Chromatography ID:CH001565
Chromatography Summary:Molecular species were resolved by reversed-phase liquid chromatography in the presence of class and sub-class specific internal standard compounds added to each sample. Selectivity was further enhanced by scheduling the detection of each compound according to its elution from the high-performance liquid chromatography (HPLC) column, known as scheduled MRM (sMRM).
Instrument Name:Waters Acquity
Column Name:Agilent Eclipse XDB-PLUS C18 (50 x 1.2mm, 1.8um)
Chromatography Type:Reversed phase

MS:

MS ID:MS001982
Analysis ID:AN002130
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:MI M+NH4
Ion Mode:POSITIVE
  
MS ID:MS001983
Analysis ID:AN002131
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:sMRM Prec 184 u
Ion Mode:POSITIVE
  
MS ID:MS001984
Analysis ID:AN002132
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:sMRM NL 141 u
Ion Mode:POSITIVE
  
MS ID:MS001985
Analysis ID:AN002133
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:sMRM Prec 241 u
Ion Mode:NEGATIVE
  
MS ID:MS001986
Analysis ID:AN002134
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:sMRM NL 172 u
Ion Mode:POSITIVE
  
MS ID:MS001987
Analysis ID:AN002135
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:sMRM NL 87 u
Ion Mode:NEGATIVE
  
MS ID:MS001988
Analysis ID:AN002136
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:sMRM Prec 184 u (KOH)
Ion Mode:POSITIVE
  
MS ID:MS001989
Analysis ID:AN002137
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:sMRM Prec SB frag.
Ion Mode:POSITIVE
  
MS ID:MS001990
Analysis ID:AN002138
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:sMRM Prec 369 u
Ion Mode:POSITIVE
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