#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