#METABOLOMICS WORKBENCH hatalbott2_20191118_161906 DATATRACK_ID:1852 STUDY_ID:ST001286 ANALYSIS_ID:AN002133 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 Prec 241 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 LPI(14:1) 2.103278237 0 0 LPI(14:0) 0.138006547 0 0 LPI(O-18:0) 0.492841999 0 0 LPI(18:1) 0 0.084024067 0.728570361 LPI(18:0) 4.549599498 0 1.164452195 LPI(20:4) 1.22817814 0 0.357535082 PI(30:1) 0.369833681 0 0.858249323 PI(32:2) 0 0.011853411 0 PI(32:1) 0 0 0 PI(34:5) 0 0.091650943 0 PI(34:4) 0 0 0 PI(34:2) 1.318730821 0.581625544 3.274412769 PI(34:1) 5.375213965 0.399600871 10.56553481 PI(34:0) 0.026927095 0.446148766 0.402882731 PI(P-36:0)/PI(O-36:1) 0 0 0 PI(36:5) 0 0 0 PI(36:4) 4.641139077 2.810002177 9.742183002 PI(36:3) 4.502299238 2.039406386 5.625851219 PI(36:2) 9.833746999 6.073976778 17.0759477 PI(36:1) 0 0 1.366491931 PI(36:0) 0.116473646 0 0.023081458 PI(P-38:1)/PI(O-38:2) 0 0 0 PI(38:6) 1.605948909 0.957849782 3.248987926 PI(38:5) 26.4969523 14.27532656 40.53489221 PI(38:4) 97.7106087 58.68040348 130.5637175 PI(38:3) 11.52807024 11.03414949 22.11165379 PI(38:2) 0.737576349 0.515908563 2.256851337 PI(38:1) 0 0 0 PI(38:0) 0 0.017959361 0 PI(40:7) 0.483732335 0.125798984 1.193164743 PI(40:6) 11.59648189 4.002056604 17.60110932 PI(40:5) 39.39430247 26.35229028 54.07495912 PI(40:4) 4.438968062 6.867571843 13.04431111 PI(40:3) 0.018771162 0.103052975 0.193880728 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 LPI(14:1) C23H43O12P 542.25 541.2 / 241.0 1.05 541.2 LPI(14:0) C23H45O12P 544.26 543.2 / 241.0 1.05 543.2 LPI(O-18:0) C27H55O11P 586.35 585.3 / 241.0 1.05 585.3 LPI(18:1) C27H51O12P 598.31 597.3 / 241.0 0.95 UGDOFRYHDCDVHD-YLMUREAQSA-N 597.3 LPI(18:0) C27H53O12P 600.33 599.3 / 241.0 1.16 MXAFDFDAIFZFET-DRPMWXDOSA-N 599.3 LPI(20:4) C29H49O12P 620.3 619.3 / 241.0 0.84 LXUGKKVCSTYZFK-YPTWYXPQSA-N 619.3 PI(30:1) C39H73O13P 780.48 779.5 / 241.0 3.4 779.5 PI(32:2) C41H75O13P 806.49 805.5 / 241.0 2.73 HMDB0009780 XVAYDAQDXIWIQD-NUHINHKHSA-N LMGP06010938 805.5 PI(32:1) C41H77O13P 808.51 807.5 / 241.0 2.94 HMDB0009797 RNXWIPLNUTUDRT-YHSIYXGOSA-N LMGP06010175 807.5 PI(34:5) C43H73O13P 828.48 827.5 / 241.0 3.36 827.5 PI(34:4) C43H75O13P 830.49 829.5 / 241.0 3.47 829.5 PI(34:2) C43H79O13P 834.53 833.5 / 241.0 3.05 HMDB0062653 BSNJSZUDOMPYIR-FJESRZELSA-N 833.5 PI(34:1) C43H81O13P 836.54 835.5 / 241.0 3.15 PDLAMJKMOKWLAJ-ZNHRTHKOSA-N 835.5 PI(34:0) C43H83O13P 838.56 837.6 / 241.0 3.15 HMDB0062582 NEXFZIYXCPIHEF-XVQBWIJQSA-N 837.6 PI(P-36:0)/PI(O-36:1) C45H87O12P 850.59 PI(36:5) C45H77O13P 856.51 855.6 / 241.0 3.05 855.6 PI(36:4) C45H79O13P 858.53 857.5 / 241.0 2.94 KIQYUSYSJTUGFZ-UVYWGIEFSA-N 857.5 PI(36:3) C45H81O13P 860.54 859.6 / 241.0 3.05 HMDB0009848 FGYIQWPIAQUVIU-AWZRONAASA-N LMGP06010318 859.6 PI(36:2) C45H83O13P 862.56 861.6 / 241.0 3.26 KZVRAFHIKMDULK-HJCWERLXSA-N 861.6 PI(36:1) C45H85O13P 864.57 863.6 / 241.0 3.36 863.6 PI(36:0) C45H87O13P 866.59 865.3 / 241.0 3.47 HMDB0009808 FQZQXPXKJFOAGE-SNXKPFKBSA-N LMGP06010008 865.3 PI(P-38:1)/PI(O-38:2) C47H89O12P 876.61 PI(38:6) C47H79O13P 882.53 881.5 / 241.0 2.94 881.5 PI(38:5) C47H81O13P 884.54 883.6 / 241.0 3.05 HMDB0009908 QRJDLXBDGZQKAL-LVRSVQNSSA-N 883.6 PI(38:4) C47H83O13P 886.56 885.6 / 241.0 3.12 RMRCTNVEDOWEQF-MQYWSVFMSA-N 885.6 PI(38:3) C47H85O13P 888.57 887.6 / 241.0 3.36 MHKWHDPIKFAACP-DAXMFMIASA-N 887.6 PI(38:2) C47H87O13P 890.59 889.6 / 241.0 3.21 WCMFLJINGZIEAR-OKJSOPGJSA-N 889.6 PI(38:1) C47H89O13P 892.6 891.6 / 241.0 3.35 891.6 PI(38:0) C47H91O13P 894.62 893.6 / 241.0 3.6 893.6 PI(40:7) C49H81O13P 908.54 907.6 / 241.0 3.05 907.6 PI(40:6) C49H83O13P 910.56 909.6 / 241.0 2.82 HMDB0009884 LJEBGLSLXVEYMV-FTIARRKHSA-N LMGP06010796 909.6 PI(40:5) C49H85O13P 912.57 911.6 / 241.0 3.26 911.6 PI(40:4) C49H87O13P 914.59 913.6 / 241.0 3.36 HMDB0009906 YMVJQMHRDDWISR-VYAQFWOSSA-N 913.6 PI(40:3) C49H89O13P 916.6 915.6 / 241.0 3.33 UETVUFSLJVFPBK-HWAFWUSFSA-N 915.6 METABOLITES_END #END