#METABOLOMICS WORKBENCH antoniop_20240705_211001 DATATRACK_ID:4985 STUDY_ID:ST003316 ANALYSIS_ID:AN005430 PROJECT_ID:PR002063 VERSION 1 CREATED_ON July 6, 2024, 3:06 pm #PROJECT PR:PROJECT_TITLE Effects of LDAH on the lipidome of oxLDL-treated mouse peritoneal macrophages PR:PROJECT_SUMMARY Lipid droplet-associated hydrolase (LDAH) has a lipase structure and high PR:PROJECT_SUMMARY affinity for lipid droplets of macrophages/foam cells. However, LDAH's functions PR:PROJECT_SUMMARY and lipid substrates remain poorly understood. In this project we treated mouse PR:PROJECT_SUMMARY peritoneal macrophages isolated from LDAH-transgenic, LDAH-deficient mice, and PR:PROJECT_SUMMARY their respective wild-type littermate controls with oxidized low-density PR:PROJECT_SUMMARY lipoprotein (oxLDL) and analyzed the effects of LDAH overexpression and LDAH PR:PROJECT_SUMMARY deficiency on their lipidome. PR:INSTITUTE Albany Medical College PR:DEPARTMENT MCP PR:LABORATORY Paul PR:LAST_NAME Paul PR:FIRST_NAME Antoni PR:ADDRESS 47 New Scotland Avenue, MC-8, albany, NY-12208 PR:EMAIL paula@amc.edu PR:PHONE 518-262-1158 #STUDY ST:STUDY_TITLE Effects of LDAH overexpression on the lipidome of oxLDL-treated mouse peritoneal ST:STUDY_TITLE macrophages ST:STUDY_SUMMARY Mouse peritoneal macrophages from LDAH-transgenic mice and wild-type littermate ST:STUDY_SUMMARY controls were harvested 5 days after aged 3% thioglycolate injection. 4–5 × ST:STUDY_SUMMARY 106 cells were plated in 60mm culture dishes, and cultured overnight in DMEM ST:STUDY_SUMMARY supplemented with 10% heat-inactivated fetal bovine serum (FBS). The media was ST:STUDY_SUMMARY replaced by DMEM-1% FBS supplemented with Hi-TBAR oxLDL (50 μg/ml, Alfa Aesar ST:STUDY_SUMMARY J65261) for 48h. ST:INSTITUTE Albany Medical College ST:DEPARTMENT MCP ST:LABORATORY Paul ST:LAST_NAME Paul ST:FIRST_NAME Antoni ST:ADDRESS 47 New Scotland Avenue, MC-8, Albany, NY-12208 ST:EMAIL paula@amc.edu ST:PHONE 518-262-1158 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 12 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN LDAH wild-type and LDAH-transgenic mice in C57BL/6 background #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - W1 Sample source:Peritoneal Macrophages | Genotype:Wild-type Treatment=oxLDL SUBJECT_SAMPLE_FACTORS - W2 Sample source:Peritoneal Macrophages | Genotype:Wild-type Treatment=oxLDL SUBJECT_SAMPLE_FACTORS - W3 Sample source:Peritoneal Macrophages | Genotype:Wild-type Treatment=oxLDL SUBJECT_SAMPLE_FACTORS - W4 Sample source:Peritoneal Macrophages | Genotype:Wild-type Treatment=oxLDL SUBJECT_SAMPLE_FACTORS - W5 Sample source:Peritoneal Macrophages | Genotype:Wild-type Treatment=oxLDL SUBJECT_SAMPLE_FACTORS - W6 Sample source:Peritoneal Macrophages | Genotype:Wild-type Treatment=oxLDL SUBJECT_SAMPLE_FACTORS - T1 Sample source:Peritoneal Macrophages | Genotype:LDAH-Transgenic Treatment=oxLDL SUBJECT_SAMPLE_FACTORS - T2 Sample source:Peritoneal Macrophages | Genotype:LDAH-Transgenic Treatment=oxLDL SUBJECT_SAMPLE_FACTORS - T3 Sample source:Peritoneal Macrophages | Genotype:LDAH-Transgenic Treatment=oxLDL SUBJECT_SAMPLE_FACTORS - T4 Sample source:Peritoneal Macrophages | Genotype:LDAH-Transgenic Treatment=oxLDL SUBJECT_SAMPLE_FACTORS - T5 Sample source:Peritoneal Macrophages | Genotype:LDAH-Transgenic Treatment=oxLDL SUBJECT_SAMPLE_FACTORS - T6 Sample source:Peritoneal Macrophages | Genotype:LDAH-Transgenic Treatment=oxLDL #COLLECTION CO:COLLECTION_SUMMARY Cells were collected, centrifuged, and washed 3x in cold PBS. Cells were CO:COLLECTION_SUMMARY immediately frozen and stored at -80C. CO:SAMPLE_TYPE Peritoneal macrophages #TREATMENT TR:TREATMENT_SUMMARY Cells were treated with Hi-TBAR oxLDL (50 μg/ml, Alfa Aesar J65261) for 48h in TR:TREATMENT_SUMMARY DMEM-1% FBS. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Monophasic lipid extracts were diluted into isopropanol: methanol (2:1,v:v) SP:SAMPLEPREP_SUMMARY containing 20 mM ammonium formate and analyzed by flow injection high SP:SAMPLEPREP_SUMMARY resolution/accurate MS and tandem MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Lipids were analyzed by flow injection high resolution/accurate MS and tandem CH:CHROMATOGRAPHY_SUMMARY MS. Lipid species were identified using the Lipid Mass Spectrum Analysis (LIMSA) CH:CHROMATOGRAPHY_SUMMARY v.1.0 software linear fit algorithm, in conjunction with a user-defined database CH:CHROMATOGRAPHY_SUMMARY of hypothetical lipid compounds for automated peak finding and correction of 13C CH:CHROMATOGRAPHY_SUMMARY isotope effects. Relative quantification of lipid abundance between samples was CH:CHROMATOGRAPHY_SUMMARY performed by normalization of target lipid ion peak areas to the di-myristoyl CH:CHROMATOGRAPHY_SUMMARY phosphatidylcholine internal standard. CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME none CH:COLUMN_NAME none CH:SOLVENT_A none CH:SOLVENT_B none CH:FLOW_GRADIENT none CH:FLOW_RATE none CH:COLUMN_TEMPERATURE none #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Velos LTQ Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Lipid sample was introduced via nanoESI to a high resolution / accurate mass MS:MS_COMMENTS Thermo Scientific model LTQ Orbitrap Velos mass spectrometer (San Jose, CA) MS:MS_COMMENTS using an Advion Triversa Nanomate nESI source (Advion, Ithaca, NY) operating MS:MS_COMMENTS with a spray voltage of 1.4 kV and a gas pressure of 0.3 psi. The ion source MS:MS_COMMENTS interface settings (inlet temperature of 100°C and S-Lens value of 50%) were MS:MS_COMMENTS optimized to maximize the sensitivity of the precursor ions while minimizing MS:MS_COMMENTS ‘in-source’ fragmentation. MS:MS_RESULTS_FILE ST003316_AN005430_Results.txt UNITS:Normalized intensity Has m/z:Yes Has RT:No RT units:No RT data #END