#METABOLOMICS WORKBENCH 378882758_20240405_203557 DATATRACK_ID:4762 STUDY_ID:ST003258 ANALYSIS_ID:AN005340 PROJECT_ID:PR002022 VERSION 1 CREATED_ON 06-14-2024 #PROJECT PR:PROJECT_TITLE Muscular Rev-erb-mediated diurnal metabolic rhythm PR:PROJECT_SUMMARY Rev-erb plays a pivotal role in coordinating circadian rhythms and in regulating PR:PROJECT_SUMMARY metabolism. However, The regulation of muscle metabolism by Rev-erbs is still PR:PROJECT_SUMMARY not fully understood. In this study, we explored the mechanisms through which PR:PROJECT_SUMMARY Rev-erb regulates skeletal muscle by using a mouse model with Rev-erbs muscle PR:PROJECT_SUMMARY knockout. PR:INSTITUTE Baylor College of Medicine PR:LAST_NAME Jidong PR:FIRST_NAME Liu PR:ADDRESS One Baylor Plaza,Houston, TX, 77030 PR:EMAIL jidong6@outlook.com PR:PHONE 7137983159 PR:DOI http://dx.doi.org/10.21228/M87V6Z #STUDY ST:STUDY_TITLE Rev-erb's Role in Muscle Metabolism: Mass Spectrometry Analysis in WT and ST:STUDY_TITLE Rev-erb skeletal muscle specific knockout Mice ST:STUDY_SUMMARY We used WT and Rev-erb skeletal muscle specific knockout mouse models to explore ST:STUDY_SUMMARY the changes in muscle metabolites at different time points, aiming to clarify ST:STUDY_SUMMARY the regulatory mechanism of Rev-erb on metabolism. The study design involved ST:STUDY_SUMMARY taking 3 mice each from the WT and muscle-specific knockout models at ZT10 and ST:STUDY_SUMMARY ZT22 (ZT, Zeitgeber Time), respectively, and performing mass spectrometry to ST:STUDY_SUMMARY detect muscle metabolites (Glycolysis, TCA, Amino acids, and ST:STUDY_SUMMARY Carnitine).Metabolomics profiling of muscles revealed robust changes in amino ST:STUDY_SUMMARY acids, glycolytic intermediates, and carnitines between KO and WT muscles, ST:STUDY_SUMMARY particularly at ZT22. ST:INSTITUTE Baylor College of Medicine ST:LAST_NAME Jidong ST:FIRST_NAME Liu ST:ADDRESS One Baylor Plaza,Houston, TX, 77030 ST:EMAIL jidong6@outlook.com ST:PHONE 7137983159 ST:SUBMIT_DATE 2024-04-05 #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 - Sa_10004 Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT10 RAW_FILE_NAME(Raw file name)=Sa_10004 SUBJECT_SAMPLE_FACTORS - Sa_10005 Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT10 RAW_FILE_NAME(Raw file name)=Sa_10005 SUBJECT_SAMPLE_FACTORS - Sa_10006 Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT10 RAW_FILE_NAME(Raw file name)=Sa_10006 SUBJECT_SAMPLE_FACTORS - Sa_10010 Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT22 RAW_FILE_NAME(Raw file name)=Sa_10010 SUBJECT_SAMPLE_FACTORS - Sa_10011 Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT22 RAW_FILE_NAME(Raw file name)=Sa_10011 SUBJECT_SAMPLE_FACTORS - Sa_10012 Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT22 RAW_FILE_NAME(Raw file name)=Sa_10012 SUBJECT_SAMPLE_FACTORS - Sa_10001 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT10 RAW_FILE_NAME(Raw file name)=Sa_10001 SUBJECT_SAMPLE_FACTORS - Sa_10002 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT10 RAW_FILE_NAME(Raw file name)=Sa_10002 SUBJECT_SAMPLE_FACTORS - Sa_10003 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT10 RAW_FILE_NAME(Raw file name)=Sa_10003 SUBJECT_SAMPLE_FACTORS - Sa_10007 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT22 RAW_FILE_NAME(Raw file name)=Sa_10007 SUBJECT_SAMPLE_FACTORS - Sa_10008 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT22 RAW_FILE_NAME(Raw file name)=Sa_10008 SUBJECT_SAMPLE_FACTORS - Sa_10009 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT22 RAW_FILE_NAME(Raw file name)=Sa_10009 #COLLECTION CO:COLLECTION_SUMMARY Snap-frozen quadriceps were collected from both WT and Rev-erb muscle-specific CO:COLLECTION_SUMMARY KO mice and mice were harvest at 8 months of age. The experiment involves CO:COLLECTION_SUMMARY collecting muscle samples at ZT10 and ZT22 (ZT, zeitgeber time) for testing. CO:SAMPLE_TYPE Muscle #TREATMENT TR:TREATMENT_SUMMARY Male mice were fed with chow diet, skeletal muscle from WT and KO collected at TR:TREATMENT_SUMMARY Zeitgeber time (ZT10) and Zeitgeber time (ZT22), respectively. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 30mg of each tissue sample was taken. To this 750 μL ice-cold methanol:water SP:SAMPLEPREP_SUMMARY (4:1) containing 20 μL spiked internal standards was added to each tissue SP:SAMPLEPREP_SUMMARY sample. Ice-cold chloroform and water were added in a 3:1 ratio for a final SP:SAMPLEPREP_SUMMARY proportion of 4:3:2 methanol:chloroform:water. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Separation of TCA and glycolysis metabolites were performed using 5 mM ammonium CH:CHROMATOGRAPHY_SUMMARY acetate in water as buffer pH 9.9 (A), and 100% acetonitrile as buffer (B) using CH:CHROMATOGRAPHY_SUMMARY Luna 3 μM NH2 column (Phenomenex, Torrance, CA) and measured 6495 Triple CH:CHROMATOGRAPHY_SUMMARY Quadrupole mass spectrometer via ESI negative mode (Agilent Technologies, Santa CH:CHROMATOGRAPHY_SUMMARY Clara, CA). The Gradient used is as follows: 0-20 min-80% B (Flow rate 0.2 CH:CHROMATOGRAPHY_SUMMARY ml/min); 20-20.10 min- 80% to 2 % B; 20.10-25 min-2% B (Flow rate 0.3 ml/min); CH:CHROMATOGRAPHY_SUMMARY 25-30 min 80% B (Flow rate 0.35 ml/min); 30-35 min-80%B (Flow rate 0.4 ml/min); CH:CHROMATOGRAPHY_SUMMARY 35-37.80 min 80% B (Flow rate 0.4 ml/min); followed by re-equilibration at the CH:CHROMATOGRAPHY_SUMMARY end of the gradient to the initial starting condition 80% B at Flow rate of 0.2 CH:CHROMATOGRAPHY_SUMMARY ml/min. CH:INSTRUMENT_NAME Agilent 6495 QQQ LC-MS systems CH:COLUMN_NAME Phenomenex Luna NH2 (150 x 2mm,3um) CH:COLUMN_TEMPERATURE 40 CH:FLOW_GRADIENT 0-20min: 80% B (Flow rate 0.2 ml/min); 20-20.10min: 80% to 2% B; 20.10-25min: 2% CH:FLOW_GRADIENT to 80% B (Flow rate 0.3 ml/min); 25-30min: 80% B (Flow rate 0.35 ml/min); CH:FLOW_GRADIENT 30-35min: 80%B (Flow rate 0.4 ml/min); 35-37.80min: 80% B (Flow rate 0.4 CH:FLOW_GRADIENT ml/min), followed by re-equilibration at the end of the gradient to the initial CH:FLOW_GRADIENT starting condition 80% B at Flow rate of 0.2 ml/min CH:FLOW_RATE 0.2 mL/min CH:SOLVENT_A 100% water; 5mM ammonium acetate CH:SOLVENT_B 100% acetonitrile CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ LC-MS systems MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:MS_COMMENTS The data were analyzed using MetaboAnalyst 6.0, which were first normalized by MS:MS_COMMENTS internal standards, log transformation, and auto-scaling methods. Statistical MS:MS_COMMENTS differences were determined using two-way ANOVA followed by Tukey test. MS:MS_COMMENTS Differential metabolites were identified by p-value < 0.05. MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples Sa_10004 Sa_10005 Sa_10006 Sa_10010 Sa_10011 Sa_10012 Sa_10001 Sa_10002 Sa_10003 Sa_10007 Sa_10008 Sa_10009 Factors Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT10 Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT10 Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT10 Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT22 Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT22 Sample source:Quadricep muscle | Genotype:Rev-erb muscle specific knock out | Zeitgeber time:ZT22 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT10 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT10 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT10 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT22 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT22 Sample source:Quadricep muscle | Genotype:wild type | Zeitgeber time:ZT22 Aspartic acid 580977.0000 1611823.0000 1924877.0000 2257500.0000 1681646.0000 3717757.0000 4472261.0000 4939173.0000 5542703.0000 1809356.0000 2405161.0000 1973994.0000 Citrate 1046613.0000 975461.0000 1005095.0000 1210769.0000 1625798.0000 1058583.0000 1240468.0000 620044.0000 1537945.0000 783216.0000 964906.0000 704623.0000 Erythrose-4-P 426361.0000 172140.0000 268622.0000 860890.0000 1063393.0000 1053368.0000 233657.0000 77962.0000 102349.0000 1360692.0000 1395372.0000 2123840.0000 Fumarate 204129.0000 321233.0000 321514.0000 286316.0000 151644.0000 367176.0000 113188.0000 200277.0000 80159.0000 205782.0000 185659.0000 304457.0000 G6P/F6P 1977434.0000 313801.0000 777305.0000 6500574.0000 28020959.0000 12578833.0000 817322.0000 441053.0000 219362.0000 6017322.0000 8189363.0000 14331982.0000 Glucose/Fructose 6808128.0000 4310308.0000 5925125.0000 5186769.0000 6020882.0000 6149398.0000 4058755.0000 3629207.0000 3528790.0000 7404192.0000 5947290.0000 7701289.0000 Glyceraldehyde 3-phosphate 13993.0000 17881.0000 17568.0000 16670.0000 28003.0000 22676.0000 11604.0000 14787.0000 4396.0000 15045.0000 15302.0000 47078.0000 Glycerol 3 Phosphate 171517.0000 315173.0000 177571.0000 164290.0000 50755.0000 87582.0000 56998.0000 41284.0000 53076.0000 584070.0000 209027.0000 984389.0000 Hydroxy glutarate 621008.0000 1162427.0000 709637.0000 1365372.0000 709359.0000 1454196.0000 1225961.0000 1251590.0000 1036170.0000 1382860.0000 973191.0000 2268886.0000 Ketoglutarate 965104.0000 761727.0000 985792.0000 1020754.0000 678510.0000 862074.0000 1402407.0000 1117383.0000 1062343.0000 1367560.0000 1100591.0000 1657429.0000 Lactate 40570296.0000 30640205.0000 33909167.0000 33565995.0000 33346891.0000 34238978.0000 29040173.0000 29610200.0000 25389120.0000 38882394.0000 43540842.0000 43800911.0000 L-Zeatine (ISTD) 1920832.0000 2045870.0000 2034917.0000 1745708.0000 1828681.0000 1915601.0000 1870041.0000 2290442.0000 1878705.0000 1908170.0000 2052442.0000 2259565.0000 Malate 8045375.0000 13001540.0000 12416290.0000 10351796.0000 6223156.0000 11318906.0000 3497666.0000 6397015.0000 2757485.0000 7277071.0000 7824047.0000 10764632.0000 Oxalate 29224.0000 35613.0000 32546.0000 28939.0000 33707.0000 39252.0000 26827.0000 31419.0000 26807.0000 31011.0000 27346.0000 33489.0000 PEP 36798.0000 30658.0000 38265.0000 66177.0000 91517.0000 117747.0000 37508.0000 27163.0000 14994.0000 95810.0000 160605.0000 233446.0000 Ribose/Ribulose/xylulose-5P 2736577.0000 3100496.0000 2811231.0000 1225033.0000 909269.0000 1160519.0000 988177.0000 1534361.0000 944579.0000 2549077.0000 1605197.0000 2495012.0000 Ribulose 5P 2674631.0000 2898078.0000 2661741.0000 1225033.0000 909269.0000 1160519.0000 909720.0000 1415231.0000 899175.0000 2442080.0000 1484395.0000 2363060.0000 Sedoheptulose-7-P 21037.0000 14401.0000 31678.0000 15816.0000 16892.0000 26402.0000 17676.0000 18245.0000 15795.0000 94637.0000 57354.0000 45878.0000 Succinate 546107.0000 991024.0000 509105.0000 447609.0000 266209.0000 269203.0000 263360.0000 515293.0000 275884.0000 409709.0000 309581.0000 685251.0000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant Aspartic acid Citrate Erythrose-4-P Fumarate G6P/F6P Glucose/Fructose Glyceraldehyde 3-phosphate Glycerol 3 Phosphate Hydroxy glutarate Ketoglutarate Lactate L-Zeatine (ISTD) Malate Oxalate PEP Ribose/Ribulose/xylulose-5P Ribulose 5P Sedoheptulose-7-P Succinate METABOLITES_END #END