#METABOLOMICS WORKBENCH hatalbott2_20191118_161906 DATATRACK_ID:1852 STUDY_ID:ST001286 ANALYSIS_ID:AN002138 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 XDB-PLUS C18 (50 x 1.2mm, 1.8um) #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 369 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 CE(14:0) 0 0 9.286732043 CE(14:1) 2.724749811 0.185013876 5.735430157 CE(15:0) 4.143669031 7.058977677 53.37635324 CE(16:0) 1.803383339 0 19.77338015 CE(16:1) 0 0 2.177393532 CE(17:0) 1.8634513 2.113999374 8.534293768 CE(17:1) 0 0 1.947847942 CE(18:0) 2.681499188 0 18.34145445 CE(18:1) 10.68211859 3.381390059 68.91887738 CE(18:2) 2.343509097 0.107924761 26.79617638 CE(18:3) 0.463972533 0 2.551848931 CE(19:0) 0 0 0 CE(20:0) 3.68765151 2.512304415 10.93072798 CE(20:1) 1.002018773 0 12.02422528 CE(20:2) 1.005010272 0.073897814 8.099200426 CE(20:3) 1.764008301 0.563296768 13.83041803 CE(20:4) 3.419667549 1.724701894 37.1405632 CE(22:0) 0 0 0 CE(22:1) 0 0 10.06141567 CE(22:4) 5.123607019 3.444202496 51.67726969 CE(22:5) 3.339898947 0.94202278 30.23036739 CE(22:6) 0.758660177 0.042943029 3.335241912 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 CE(14:0) C41H72O2 596.55 34670 HMDB0006725 SJDMTGSQPOFVLR-ZPQCIJQQSA-N LMST01020004 CE(14:1) C41H70O2 594.54 34687 HMDB0062458 LAMGDJMPDNVWTB-BJZZQDJASA-N LMST01020021 CE(15:0) C42H74O2 610.57 34693 HMDB0060057 BDBPUUADYAQHAV-LNVIZZJGSA-N LMST01020027 CE(16:0) C43H76O2 624.58 34671 HMDB0000885 BBJQPKLGPMQWBU-JADYGXMDSA-N LMST01020005 CE(16:1) C43H74O2 622.57 HODJWNWCVNUPAQ-XDOSKZMUSA-N LMST01020006 CE(17:0) C44H78O2 638.60 34692 HMDB0060059 PPQNZVDOBYGOLY-QEXSOPRKSA-N LMST01020026 CE(17:1) C44H76O2 636.58 34689 HMDB0062454 RLMIGWIAENJHMP-RJRTUNKTSA-N LMST01020023 CE(18:0) C45H80O2 652.62 34673 HMDB0062461 XHRPOTDGOASDJS-XNTGVSEISA-N LMST01020007 CE(18:1) C45H78O2 650.60 34669 HMDB0000918 RJECHNNFRHZQKU-RMUVNZEASA-N LMST01020003 CE(18:2) C45H76O2 648.58 34674 HMDB0000610 NAACPBBQTFFYQB-LJAITQKLSA-N LMST01020008 CE(18:3) C45H74O2 646.57 34675 HMDB0010370 FYMCIBHUFSIWCE-WVXFKAQASA-N LMST01020009 CE(19:0) C46H82O2 666.63 HMDB0006738 VHYWECIJXTVRSG-TVDLSCFRSA-N CE(20:0) C47H84O2 680.65 34676 HMDB0062459 SUOVMGLZSOAHJY-JREUTYQLSA-N LMST01020010 CE(20:1) C47H82O2 678.63 34677 OWTYWMJVQZQWFH-UQPFUGTCSA-N LMST01020011 CE(20:2) C47H80O2 676.62 34678 HMDB0062455 REFJKOQDWJELKE-AKLGJIFXSA-N LMST01020012 CE(20:3) C47H78O2 674.60 40920 MLPRJPSMAFZPLA-BBFGHUFCSA-N LMST01020013 CE(20:4) C47H76O2 672.58 HMDB0006726 IMXSFYNMSOULQS-BEDFLICRSA-N LMST01020014 CE(22:0) C49H88O2 708.68 34682 WBOQXYUYHINMOC-FTAWAYKBSA-N LMST01020016 CE(22:1) C49H86O2 706.66 34691 HMDB0010372 SQHUGNAFKZZXOT-JWTURFAQSA-N CE(22:4) C49H80O2 700.62 34684 HMDB0006729 ITGTXSFLBABXQA-BMURBKCOSA-N LMST01020018 CE(22:5) C49H78O2 698.60 40921 HMDB0062457 XOLZNHXNFMEUGA-DCDLNIKOSA-N LMST01020031 CE(22:6) C49H76O2 696.58 34685 VOEVEGPMRIYYKC-HNJOWPRISA-N LMST01020019 METABOLITES_END #END