#METABOLOMICS WORKBENCH 378882758_20240405_203557 DATATRACK_ID:4762 STUDY_ID:ST003258 ANALYSIS_ID:AN005341 PROJECT_ID:PR002022 VERSION 1 CREATED_ON June 13, 2024, 9:37 pm #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 #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 #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]Raw file names and additional sample data 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_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_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 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 #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 and measurement of amino acids and carnitines were performed using CH:CHROMATOGRAPHY_SUMMARY Zorbax eclipse XDB C18, 1.8 micron, 4.6 x 100 mm column on 6495 triple CH:CHROMATOGRAPHY_SUMMARY quadrupole mass spectrometer via ESI Positive mode (Agilent Technologies, Santa CH:CHROMATOGRAPHY_SUMMARY Clara, CA). Mobile phases A and B were 0.1% formic acid in water and CH:CHROMATOGRAPHY_SUMMARY acetonitrile, respectively. The gradient used is as follows: 0-6 min - 2% of B, CH:CHROMATOGRAPHY_SUMMARY 6-6.50 min-30 % B, 6.50-7 min- 90% of B, 7-12 min 95% of B,13 min 2% of B CH:CHROMATOGRAPHY_SUMMARY followed by re-equilibration at the end of the gradient 20 min to the initial CH:CHROMATOGRAPHY_SUMMARY starting condition 2% of B. Flow rate: 0.2 ml/min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 6495 QQQ LC-MS systems CH:COLUMN_NAME Agilent ZORBAX Eclipse XDB-C18 (100 x 4.6mm,1.8um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0-6 min - 2% of B, 6-6.50 min-30 % B, 6.50-7 min- 90% of B, 7-12 min 95% of B,13 CH:FLOW_GRADIENT min 2% of B followed by re-equilibration at the end of the gradient 20 min to CH:FLOW_GRADIENT the initial starting condition 2% of B. CH:FLOW_RATE 0.2mL/min CH:COLUMN_TEMPERATURE 40 #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:ION_MODE POSITIVE 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_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples Sa_10001 Sa_10002 Sa_10003 Sa_10004 Sa_10005 Sa_10006 Sa_10007 Sa_10008 Sa_10009 Sa_10010 Sa_10011 Sa_10012 Factors 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: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: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 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 L-Tesrosterone (ISTD) 36009 30206 44646 35835 30779 26679 45262 39552 48017 31732 33757 36221 Alanine 40096325 38793844 44220106 45818653 42006222 45576481 42985141 40379509 45372109 47695399 48422675 52656844 Arginine 5790033 13306610 8048589 16192917 7387034 9182004 49555178 44248679 36328751 10930536 15819160 32656195 Asparagine 1338883 1830900 3067762 3314051 1588456 2138857 5010375 5313780 5049194 7907207 5315329 11029006 Glutamic acid 857964 1520698 1570434 833798 1415472 1434880 967769 1609091 1393515 1136434 2202395 1632913 Glutamine 2993411 5661005 3369176 4192030 2439551 2766720 26662750 33918229 23036742 5419861 6083119 13195265 Histidine 4600603 4253030 5732188 3893732 5747896 6564508 4725647 4328156 5044013 4603815 4280340 5672095 Homoserine 167493 268731 322871 264194 167551 228340 310192 504293 340913 513229 776415 983108 Isoleucine 564698047 553553523 616721591 551543629 605820118 580408636 653441686 699710316 553523851 628765595 606276386 695015509 Lysine 3703873 6930529 4135256 5292205 2976377 5072517 42068315 37995554 26677888 11256060 12900300 25758099 Methionine 13748822 13541292 10832758 19895088 18872826 15263255 55578727 51983235 35884404 56882385 42574898 61524087 Ornithine 1665276 1742171 2374846 1774190 1962647 2112155 1451645 2192539 1824179 1892510 1745245 2144445 Phenyl alanine 494529251 450421283 490181585 583679911 571711335 546380581 685450414 739094932 516910003 672317028 606405927 786863998 Proline 24544835 28018858 32014417 28148662 21841494 28382518 40564599 37396759 58263745 33788791 32631312 64826876 Serine 41173 69846 66437 55566 29052 31635 38079 40810 34353 69618 47522 89442 Threonine 154327 268731 322871 173338 124653 228340 310192 504293 340913 513229 776415 983108 Tryptophan 66160603 69376407 122801573 85962214 78935409 100265325 114615290 129758291 201443029 140036681 155631089 155102664 Tyrosine 34180966 34681710 38308152 48417082 54459574 42308222 75485500 110700815 82358528 106621653 88731504 81929053 Valine 41088122 43388313 62168783 36355030 38680415 46503300 39982250 39826883 43442785 47146184 54521970 56192827 L-Zeatine (ISTD) 31190366 31551916 32819349 40556198 35631865 38111193 40136077 37200410 39902196 39166076 36466681 42609837 2-Methylbutyrylcarnitine 2364619 2829631 4192902 5192813 9444734 4898926 22670585 21943604 6501025 11721724 12738431 19140266 Acetyl Carnitine 331228506 285130558 303697958 301454388 231832324 314915255 464555788 640839764 475126983 319249394 227739164 308358140 Butyrylcarnitine 56934789 79798031 88365405 134109724 189144834 150776695 192832029 350359971 146566741 187005983 113443156 241826470 Carnitine 20462778 29091147 16846688 18322623 12348277 12873691 30185382 28756510 21050631 16550873 10889408 16993157 Deoxy carnitine 3438804 4207300 3241673 5529236 3184322 3780424 3743721 4181651 4204164 3816159 2835912 8649132 Heptanoyl Carnitine 22614513 30355403 39202035 49244664 65089836 76948975 25926345 34966439 54677795 34730143 13823215 28219508 Isobutyryl Carnitine 53581551 80603533 77556833 127431524 177215523 169308415 181576900 318946022 152734188 179908506 116965776 245950077 Isovaleryl Carnitine 13949815 15877205 25730481 29663619 55165957 35138018 164349737 127581462 35728950 65564746 84906002 121822717 MethylGlutaryl carnitine 29953 35018 22577 24354 39056 50185 33100 38596 46051 27144 29363 23237 Octanoylcarnitine 11776613 15971643 17052254 19197545 28186778 29722915 11716606 13143249 24416226 17027287 9132723 15927553 Palmitoylcarnitine 2068727 912983 916116 451328 1209088 930608 495688 754517 863689 437550 353624 661674 Propionyl Carnitine 15869469 24592620 24404532 31561457 24418175 27911655 52117751 58828053 32408003 30486711 16693828 27946924 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name L-Tesrosterone (ISTD) Alanine Arginine Asparagine Glutamic acid Glutamine Histidine Homoserine Isoleucine Lysine Methionine Ornithine Phenyl alanine Proline Serine Threonine Tryptophan Tyrosine Valine L-Zeatine (ISTD) 2-Methylbutyrylcarnitine Acetyl Carnitine Butyrylcarnitine Carnitine Deoxy carnitine Heptanoyl Carnitine Isobutyryl Carnitine Isovaleryl Carnitine MethylGlutaryl carnitine Octanoylcarnitine Palmitoylcarnitine Propionyl Carnitine METABOLITES_END #END