#METABOLOMICS WORKBENCH donglianglu_20240319_191948 DATATRACK_ID:4728 STUDY_ID:ST003142 ANALYSIS_ID:AN005155 PROJECT_ID:PR001953 VERSION 1 CREATED_ON March 21, 2024, 5:54 pm #PROJECT PR:PROJECT_TITLE Global Lipidomics of Serum Samples from control and ACOX1-LKO Mouse PR:PROJECT_SUMMARY In this study, we use a Acox1 liver specific knock mouse to explore the role of PR:PROJECT_SUMMARY liver peroxisomal fatty acids beta-oxidation in whole body metabolic PR:PROJECT_SUMMARY homeostasis. As the key enzyme of peroxisomal fatty acid beta-oxidation, knock PR:PROJECT_SUMMARY out Acox1 in liver will affect the very long chain fatty acid accoumaltion in PR:PROJECT_SUMMARY liver and their secretion into circulating. PR:INSTITUTE Washington University School of Medicine PR:LAST_NAME Lu PR:FIRST_NAME Dongliang PR:ADDRESS 660 S. Euclid Ave., St. Louis, Missouri, 63110, USA PR:EMAIL ludong-liang@wustl.edu PR:PHONE 3147476766 #STUDY ST:STUDY_TITLE Effect of liver Acox1 knockout on serum lipidome in mice ST:STUDY_SUMMARY The liver gene expression of the peroxisomal β-oxidation enzyme acyl-coenzyme A ST:STUDY_SUMMARY oxidase 1 (ACOX1), which catabolizes very long chain fatty acids (VLCFA), ST:STUDY_SUMMARY increases in the context of obesity. To check if liver peroxisomal fatty acids ST:STUDY_SUMMARY beta-oxidation deficiency will affect whole body metabolic homeostasis through ST:STUDY_SUMMARY circulating lipids. We analyzed serum samples from 5 WT and 5 Acox1-LKO mice. ST:INSTITUTE Washington University in St. Louis ST:LAST_NAME Lu ST:FIRST_NAME Dongliang ST:ADDRESS 660 S. Euclid Ave. ST:EMAIL ludong-liang@wustl.edu ST:PHONE 3147476766 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - Sample1 Sample_ID:Wild-type | Sample source:mouse serum | Sample source:- SUBJECT_SAMPLE_FACTORS - Sample2 Sample_ID:Wild-type | Sample source:mouse serum | Sample source:- SUBJECT_SAMPLE_FACTORS - Sample3 Sample_ID:Wild-type | Sample source:mouse serum | Sample source:- SUBJECT_SAMPLE_FACTORS - Sample4 Sample_ID:Wild-type | Sample source:mouse serum | Sample source:- SUBJECT_SAMPLE_FACTORS - Sample5 Sample_ID:Wild-type | Sample source:mouse serum | Sample source:- SUBJECT_SAMPLE_FACTORS - Sample6 Sample_ID:Acox1-LKO | Sample source:mouse serum | Sample source:- SUBJECT_SAMPLE_FACTORS - Sample7 Sample_ID:Acox1-LKO | Sample source:mouse serum | Sample source:- SUBJECT_SAMPLE_FACTORS - Sample8 Sample_ID:Acox1-LKO | Sample source:mouse serum | Sample source:- SUBJECT_SAMPLE_FACTORS - Sample9 Sample_ID:Acox1-LKO | Sample source:mouse serum | Sample source:- SUBJECT_SAMPLE_FACTORS - Sample10 Sample_ID:Acox1-LKO | Sample source:mouse serum | Sample source:- #COLLECTION CO:COLLECTION_SUMMARY After collecting the whole blood of mice, allow the blood to clot by leaving at CO:COLLECTION_SUMMARY room temperature for 30 minutes. Then serum were collected by centrifugation at CO:COLLECTION_SUMMARY 2000 g for 10 minutes, and save at -80C CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY WT and Acox1-LKO mice were maintained under constant temperature (24°C), TR:TREATMENT_SUMMARY circulating air and humidity (45-65%) with 12h:12h light/dark cycle. All mice TR:TREATMENT_SUMMARY had free access to chew diet and water. 10 weeks age mice were used for TR:TREATMENT_SUMMARY analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The lipids extraction was performed strictly following the SOP established based SP:SAMPLEPREP_SUMMARY on a modified Folch liquid-liquid extraction protocol. Briefly, an aliquot of SP:SAMPLEPREP_SUMMARY 4.5 μL of each sample was vortexed with 1.5 μL of internal standard solution SP:SAMPLEPREP_SUMMARY and methanol, followed by adding dichloromethane and vortexing for another 20 s. SP:SAMPLEPREP_SUMMARY A clean-up step was performed with water and 10 seconds of vortex. Samples were SP:SAMPLEPREP_SUMMARY allowed to equilibrate at room temperature for 10 min and centrifuged at 16,000 SP:SAMPLEPREP_SUMMARY g for 10 min at 4°C. An aliquot of the organic layer was evaporated to dryness SP:SAMPLEPREP_SUMMARY with a nitrogen blowdown evaporator. The residue was re-suspended in 4.5 μL of SP:SAMPLEPREP_SUMMARY NovaMT MixB, vortexed for 1 min, and diluted with 40.5 μL of NovaMT MixA #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 10 mM NH4COOH in 50:40:10 MeOH/ACN/Water CH:SOLVENT_B 10 mM NH4COOH in 95:5 IPA/Water CH:FLOW_GRADIENT t = 0 min, 5% B; t = 10 min, 40% B; t = 18.8 min, 98% B; t = 20.5 min, 98% B. CH:FLOW_RATE 250 μL/min. CH:COLUMN_TEMPERATURE 42 °C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Impact HD MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The acquisition rate was 1.44 Hz for MS acquisition and 4 – 10 Hz for MS/MS MS:MS_COMMENTS spectra acquisition, with an m/z range from 150 to 1500. For both positive and MS:MS_COMMENTS negative ionization. Intensity threshold:2500 cts for positive ionization. Lipid MS:MS_COMMENTS features were extracted and aligned using software LipidScreener 1.1.0 MS:CAPILLARY_VOLTAGE 4200 V MS:COLLISION_ENERGY 10-70 eV MS:DRY_GAS_FLOW 5.0 L/min MS:DRY_GAS_TEMP 240°C MS:NEBULIZER 1.2 Bar MS:MS_RESULTS_FILE ST003142_AN005155_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END