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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001286 |
| Study Title | Lipid composition of isolated lipid droplets from the functional bovine corpus luteum |
| Study Type | Lipidomics |
| Study 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 |
| jsdavis@unmc.edu | |
| Phone | 402-559-9079 |
| Submit Date | 2019-11-18 |
| Num Groups | 1 |
| Total Subjects | 3 |
| Num Females | 3 |
| Analysis Type Detail | LC-MS |
| Release Date | 2020-05-18 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
|---|---|---|
| SA093605 | bovine_CL_LD_replicate3 | Control |
| SA093606 | bovine_CL_LD_replicate2 | Control |
| SA093607 | bovine_CL_LD_replicate1 | Control |
| 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 |
