#METABOLOMICS WORKBENCH jsan4d_20221207_100535 DATATRACK_ID:3637 STUDY_ID:ST002396 ANALYSIS_ID:AN003903 VERSION 1 CREATED_ON 12-12-2022 #PROJECT PR:PROJECT_TITLE p53 K316P mouse liver lipidomics analysis PR:PROJECT_SUMMARY Mouse livers were isolated from four wild type (WT) and four p53 K316P (M) mice PR:PROJECT_SUMMARY for lipidomic analysis. Samples were isolated and flash frozen in liquid PR:PROJECT_SUMMARY nitrogen. Lipids were then extracted from each liver sample and analyzed using PR:PROJECT_SUMMARY mass spectrometry. PR:INSTITUTE University of North Carolina PR:LAST_NAME Sanford PR:FIRST_NAME Jack PR:ADDRESS 450 West Drive, Chapel Hill, NC, 27514 PR:EMAIL jsan4d@email.unc.edu PR:PHONE 3019284726 PR:DOI http://dx.doi.org/10.21228/M87T4V #STUDY ST:STUDY_TITLE p53 K316P mutation results in increased liver triglyceride levels and increased ST:STUDY_TITLE rates of de novo lipogenesis. ST:STUDY_SUMMARY Our lab generated the p53 K316P mouse, which mimicks a common amino acid change ST:STUDY_SUMMARY found in bats. The K316P mutation, found in the nuclear localization signal of ST:STUDY_SUMMARY p53, results in increased cytoplasmic localization of p53. We found that K316P ST:STUDY_SUMMARY mutant mice develop several metabolic phenotypes, including increased body fat ST:STUDY_SUMMARY percentage, and increased liver lipid levels. In order to determine the ST:STUDY_SUMMARY mechanism through which K316P mutation increases liver lipid levels, we ST:STUDY_SUMMARY performed metabolomic analysis of mouse livers from WT and K316P mutant mice. ST:STUDY_SUMMARY Mouse livers were isolated from four wild type (WT) and four p53 K316P (M) mice ST:STUDY_SUMMARY for lipidomic analysis. Samples were isolated and flash frozen in liquid ST:STUDY_SUMMARY nitrogen. Lipids were then extracted from each liver sample and analyzed using ST:STUDY_SUMMARY mass spectrometry. ST:INSTITUTE University of North Carolina ST:LAST_NAME Sanford ST:FIRST_NAME Jack ST:ADDRESS 450 West Drive, Chapel Hill, NC, 27514 ST:EMAIL jsan4d@email.unc.edu ST:PHONE 3019284726 ST:SUBMIT_DATE 2022-12-07 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Wild type mouse 2 WT-21RL experimental factor(s):p53 +/+ RAW_FILE_NAME=WT-21RL.mzXML SUBJECT_SAMPLE_FACTORS Wild type mouse 3 WT-22R experimental factor(s):p53 +/+ RAW_FILE_NAME=WT-22R.mzXML SUBJECT_SAMPLE_FACTORS Wild type mouse 4 WT-24R experimental factor(s):p53 +/+ RAW_FILE_NAME=WT-24R.mzXML SUBJECT_SAMPLE_FACTORS Wild type mouse 1 WT-8RL experimental factor(s):p53 +/+ RAW_FILE_NAME=WT-8RL.mzXML SUBJECT_SAMPLE_FACTORS Mutant mouse 3 m-12L experimental factor(s):p53 K316P m/m RAW_FILE_NAME=m-12L.mzXML SUBJECT_SAMPLE_FACTORS Mutant mouse 4 m-41RL experimental factor(s):p53 K316P m/m RAW_FILE_NAME=m-41RL.mzXML SUBJECT_SAMPLE_FACTORS Mutant mouse 1 m-6L experimental factor(s):p53 K316P m/m RAW_FILE_NAME=m-6L.mzXML SUBJECT_SAMPLE_FACTORS Mutant mouse 2 m-6R experimental factor(s):p53 K316P m/m RAW_FILE_NAME=m-6R.mzXML #COLLECTION CO:COLLECTION_SUMMARY Mouse liver samples were collected from mice and immediately flash frozen in CO:COLLECTION_SUMMARY liquid nitrogen. Frozen tissue samples were crushed using a morter and pestle on CO:COLLECTION_SUMMARY dry ice to homogenize tissue prior to lipid extraction. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY Male mice were C57BL/6J background, kept on a 12hr day/night cycle. Mice were TR:TREATMENT_SUMMARY 3-4 months old at the time of tissue isolation. Four littermate pairs were TR:TREATMENT_SUMMARY analyzed. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Mouse liver sections were weighed into Eppendorf tubes. The liver tissue was SP:SAMPLEPREP_SUMMARY then mashed with a spatula prior to extraction. The samples were extracted using SP:SAMPLEPREP_SUMMARY a liquid liquid partition with water (250 µL), methanol (300 µL), and MTBE (1 SP:SAMPLEPREP_SUMMARY mL). Avanti’s deuterated lipid mix, Equisplash, was used as an internal SP:SAMPLEPREP_SUMMARY standard. This was spiked into the methanol at 1.5 µg/mL and used for SP:SAMPLEPREP_SUMMARY extraction. The extracts were centrifuged at 20,000 rcf for 10 min. The top SP:SAMPLEPREP_SUMMARY layer was removed, dried down, and reconstituted in 1 mL of IPA for analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Analysis was performed using a Thermo Q Exactive Plus coupled to a Waters CH:CHROMATOGRAPHY_SUMMARY Acquity H-Class LC. A 100 mm x 2.1 mm, 1.7 µm Waters BEH C18 column was used CH:CHROMATOGRAPHY_SUMMARY for separations. The following mobile phases were used: A- 60/40 ACN/H20 B- CH:CHROMATOGRAPHY_SUMMARY 90/10 IPA/ACN; both mobile phases had 10 mM Ammonium Formate and 0.1% Formic CH:CHROMATOGRAPHY_SUMMARY Acid. A flow rate of 0.2 mL/min was used. Starting composition was 32% B, which CH:CHROMATOGRAPHY_SUMMARY increased to 40% B at 1 min (held until 1.5 min) then 45% B at 4 minutes. This CH:CHROMATOGRAPHY_SUMMARY was increased to 50% B at 5 min, 60% B at 8 min, 70% B at 11 min, and 80% B at CH:CHROMATOGRAPHY_SUMMARY 14 min (held until 16 min). At 16 min the composition switched back to starting CH:CHROMATOGRAPHY_SUMMARY conditions (32% B) and was held for 4 min to re-equilibrate the column. Samples CH:CHROMATOGRAPHY_SUMMARY were analyzed in positive/negative switching ionization mode with top 5 data CH:CHROMATOGRAPHY_SUMMARY dependent fragmentation. CH:METHODS_FILENAME Sanford_2022_Lipidomics_protocol.docx CH:INSTRUMENT_NAME Waters Acquity H-Class CH:COLUMN_NAME Waters Acquity BEH C18 (100 x 2.1mm, 1.7um) CH:FLOW_GRADIENT Starting composition was 32% B, which increased to 40% B at 1 min (held until CH:FLOW_GRADIENT 1.5 min) then 45% B at 4 minutes. This was increased to 50% B at 5 min, 60% B at CH:FLOW_GRADIENT 8 min, 70% B at 11 min, and 80% B at 14 min (held until 16 min). At 16 min the CH:FLOW_GRADIENT composition switched back to starting conditions (32% B) and was held for 4 min CH:FLOW_GRADIENT to re-equilibrate the column CH:FLOW_RATE 0.2 ml/min CH:SOLVENT_A 60% acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium formate CH:SOLVENT_B 90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Sanford_2022_Lipidomics_protocol.docx #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS Analysis was performed using a Thermo Q Exactive Plus coupled to a Waters MS:MS_COMMENTS Acquity H-Class LC. A 100 mm x 2.1 mm, 2.1 µm Waters BEH C18 column was used MS:MS_COMMENTS for separations. The following mobile phases were used: A- 60/40 ACN/H20 B- MS:MS_COMMENTS 90/10 IPA/ACN; both mobile phases had 10 mM Ammonium Formate and 0.1% Formic MS:MS_COMMENTS Acid. A flow rate of 0.2 mL/min was used. Starting composition was 32% B, which MS:MS_COMMENTS increased to 40% B at 1 min (held until 1.5 min) then 45% B at 4 minutes. This MS:MS_COMMENTS was increased to 50% B at 5 min, 60% B at 8 min, 70% B at 11 min, and 80% B at MS:MS_COMMENTS 14 min (held until 16 min). At 16 min the composition switched back to starting MS:MS_COMMENTS conditions (32% B) and was held for 4 min to re-equilibrate the column. Samples MS:MS_COMMENTS were analyzed in positive/negative switching ionization mode with top 5 data MS:MS_COMMENTS dependent fragmentation. MS:ION_MODE UNSPECIFIED MS:MS_RESULTS_FILE ST002396_AN003903_Results.txt UNITS:peak area Has m/z:Yes #END