#METABOLOMICS WORKBENCH hatalbott2_20191118_161906 DATATRACK_ID:1852 STUDY_ID:ST001286 ANALYSIS_ID:AN002134 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 NL 172 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 LPG(12:0) 7.955668776 4.189624963 5.304808691 LPG(14:0) 0 0 0.015383986 LPG(16:1) 0.40777134 0.076105984 0.263461407 LPG(16:0) 0.017398157 0.005217155 0.048232214 LPG(18:3) 0.017051837 0.015173277 0.081403721 LPG(18:2) 0.045904096 0 0.000172457 LPG(18:1) 0.017097347 0 0.082475972 LPG(18:0) 0.043912754 0 0 LPG(20:4) 0 0.004103634 0 PG(34:1) 0.679095991 0.569547906 3.27029883 PG(36:5) 0.012995673 0.016015743 0 PG(36:4) 0.209989639 0.893036125 1.850366124 PG(36:3) 1.15254818 0 2.470817463 PG(36:2) 2.651729913 3.682584229 15.28976221 PG(36:1) 0.248312566 0.665234906 1.908526714 PG(38:6) 0.170129779 0.335999904 0.156902561 PG(38:5) 0.326950588 0.428543014 1.846450618 PG(38:4) 0 0.32878794 1.782298548 PG(38:3) 1.196754182 1.25064556 0.71752625 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 LPG(12:0) C18H37O9P 428.22 429.3 / 257.3 IACXMECMZISNLR-DLBZAZTESA-N LMGP04050011 429.3 LPG(14:0) C20H41O9P 456.25 457.3 / 285.3 LUTDZDAPSDZVAL-RBUKOAKNSA-N LMGP04050012 457.3 LPG(16:1) C22H43O9P 482.26 483.3 / 311.3 483.3 LPG(16:0) C22H45O9P 484.28 485.3 / 313.3 BVJSKAUUFXBDOB-LEWJYISDSA-N LMGP04050008 485.3 LPG(18:3) C24H43O9P 506.26 507.3 / 335.3 507.3 LPG(18:2) C24H45O9P 508.28 509.3 / 337.3 509.3 LPG(18:1) C24H47O9P 510.3 511.3 / 339.3 FQQQKGAFQIIGLQ-SNZQZGEVSA-N LMGP04050006 511.3 LPG(18:0) C18H34O 266.26 513.3 / 341.3 HFJVKBVEKQHVTO-XZOQPEGZSA-N LMGP04050009 513.3 LPG(20:4) C26H45O9P 532.28 531.4 / 359.4 GXPOZJZFGYCEMT-VEKMRQFKSA-N LMGP04050010 531.4 PG(34:1) C40H77O10P 748.53 749.4 / 577.4 ADYWCMPUNIVOEA-GPJPVTGXSA-N 749.4 PG(36:5) C42H73O10P 768.49 769.5 / 597.5 HMDB0010680 XAUQEXCGLQPOMJ-MLMWWJLQSA-N LMGP04010408 769.5 PG(36:4) C42H75O10P 770.51 771.5 / 599.5 HMDB0010580 FBAPNCMXWMGHJY-NYWCEQGFSA-N LMGP04010036 771.5 PG(36:3) C42H77O10P 772.53 773.5 / 601.5 YQISKVDQGRCZBE-WQQWEMLQSA-N LMGP04010904 773.5 PG(36:2) C42H79O10P 774.54 775.5 / 603.5 HMDB0010634 DSNRWDQKZIEDDB-SQYFZQSCSA-N LMGP04010985 775.5 PG(36:1) C42H81O10P 776.56 777.6 / 605.6 HMDB0010604 ZEFGRNLJASLRBZ-QIJYXWHJSA-N LMGP04010037 777.6 PG(38:6) C44H75O10P 794.51 795.6 / 623.6 HMDB0010669 PGUFYFFTFKQUSX-FHUCRLRLSA-N LMGP04010388 795.6 PG(38:5) C44H77O10P 796.53 797.6 / 625.6 FATXSYDWISDRSW-WFYRZQIBSA-N 797.6 PG(38:4) C44H79O10P 798.54 799.6 / 627.6 GGSONEYZZWVXFY-QPQYMHNXSA-N LMGP04010039 799.6 PG(38:3) C44H81O10P 800.56 801.7 / 629.7 HMDB0010609 YDMMRUHKIIMRLH-MNQIWUGTSA-N LMGP04010885 801.7 METABOLITES_END #END