#METABOLOMICS WORKBENCH xliu68_20220414_115257_mwtab.txt DATATRACK_ID:3201 STUDY_ID:ST002152 ANALYSIS_ID:AN003525 PROJECT_ID:PR001365 VERSION 1 CREATED_ON April 25, 2022, 9:33 am #PROJECT PR:PROJECT_TITLE Metabolomics analysis of mouse liver with or without SIRT5 deficiency PR:PROJECT_SUMMARY Lysine succinylation is a post-translational modification that has been PR:PROJECT_SUMMARY implicated in the regulation of various metabolic pathways. However, the PR:PROJECT_SUMMARY biological relevance of lysine succinylation remains largely uncertain due to PR:PROJECT_SUMMARY methodological difficulties in determining high-impact succinylation sites. In PR:PROJECT_SUMMARY the present study, multiple high stoichiometry lysine sites were identified in PR:PROJECT_SUMMARY argininosuccinate synthase (ASS1), a key enzyme in urea cycle, are regulated by PR:PROJECT_SUMMARY SIRT5. Metabolomics profiling confirms that SIRT5 deficiency decreases urea PR:PROJECT_SUMMARY cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia PR:PROJECT_SUMMARY tolerance, and reduces locomotor and exploratory activity in male mice upon PR:PROJECT_SUMMARY high-ammonium diet (HAD) feeding. Therefore, lysine succinylation plays a PR:PROJECT_SUMMARY functionally important role in ammonia metabolism. PR:INSTITUTE North Carolina State University PR:LAST_NAME Liu PR:FIRST_NAME Xiaojing PR:ADDRESS Polk Hall, RM 128 PR:EMAIL xliu68@ncsu.edu PR:PHONE 9195154387 #STUDY ST:STUDY_TITLE Metabolomics analysis of mouse liver with or without SIRT5 deficiency ST:STUDY_SUMMARY 8 Wild-type (WT) and 8 Sirt5-/- (SIRT5 KO) mice on 129 background were ST:STUDY_SUMMARY maintained on a standard chow diet (Teklad Global 18% Protein Rodent Diet, ST:STUDY_SUMMARY ENVIGO, Cat.#2018) until they were put on euthanized. Liver metabolites were ST:STUDY_SUMMARY extracted (5-10 mg) were extracted using 80% methanol/water as the extraction ST:STUDY_SUMMARY solvent. Metabolite extract was split into two tubes (one for polar metabolite ST:STUDY_SUMMARY analysis and the other one for acyl-CoA analysis), and then extraction solvent ST:STUDY_SUMMARY was evaporated using a speed vacuum concentrator. Dry pellets were stored in ST:STUDY_SUMMARY −80 °C freezer until ready for LC-MS analysis. For acyl-CoA analysis, dry ST:STUDY_SUMMARY pellets were reconstituted into 30 μL of sample solvent (water containing 50 mM ST:STUDY_SUMMARY ammonium acetate) per 6 mg tissue, and 8 μL was injected into the LC-MS. For ST:STUDY_SUMMARY non-acyl-CoA polar metabolite analysis, pellets were reconstituted into 30 μL ST:STUDY_SUMMARY of sample solvent (water:methanol:acetonitrile, 2:1:1, v/v/v) per 3 mg tissue, ST:STUDY_SUMMARY and 3 μL was injected into the LC-MS. ST:INSTITUTE North Carolina State University ST:LAST_NAME Liu ST:FIRST_NAME Xiaojing ST:ADDRESS Polk Hall, RM 128 ST:EMAIL xliu68@ncsu.edu ST:PHONE 9195154387 #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 - WT_1.raw Sample Source:Wild type RAW_FILE_NAME=WT_1.raw SUBJECT_SAMPLE_FACTORS - WT_2.raw Sample Source:Wild type RAW_FILE_NAME=WT_2.raw SUBJECT_SAMPLE_FACTORS - WT_3.raw Sample Source:Wild type RAW_FILE_NAME=WT_3.raw SUBJECT_SAMPLE_FACTORS - WT_4.raw Sample Source:Wild type RAW_FILE_NAME=WT_4.raw SUBJECT_SAMPLE_FACTORS - WT_5.raw Sample Source:Wild type RAW_FILE_NAME=WT_5.raw SUBJECT_SAMPLE_FACTORS - WT_6.raw Sample Source:Wild type RAW_FILE_NAME=WT_6.raw SUBJECT_SAMPLE_FACTORS - WT_7.raw Sample Source:Wild type RAW_FILE_NAME=WT_7.raw SUBJECT_SAMPLE_FACTORS - WT_8.raw Sample Source:Wild type RAW_FILE_NAME=WT_8.raw SUBJECT_SAMPLE_FACTORS - KO_1.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_1.raw SUBJECT_SAMPLE_FACTORS - KO_2.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_2.raw SUBJECT_SAMPLE_FACTORS - KO_3.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_3.raw SUBJECT_SAMPLE_FACTORS - KO_4.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_4.raw SUBJECT_SAMPLE_FACTORS - KO_5.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_5.raw SUBJECT_SAMPLE_FACTORS - KO_6.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_6.raw SUBJECT_SAMPLE_FACTORS - KO_7.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_7.raw SUBJECT_SAMPLE_FACTORS - KO_8.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_8.raw SUBJECT_SAMPLE_FACTORS - WT_1_luna.raw Sample Source:Wild type RAW_FILE_NAME=WT_1_luna.raw SUBJECT_SAMPLE_FACTORS - WT_2_luna.raw Sample Source:Wild type RAW_FILE_NAME=WT_2_luna.raw SUBJECT_SAMPLE_FACTORS - WT_3_luna.raw Sample Source:Wild type RAW_FILE_NAME=WT_3_luna.raw SUBJECT_SAMPLE_FACTORS - WT_4_luna.raw Sample Source:Wild type RAW_FILE_NAME=WT_4_luna.raw SUBJECT_SAMPLE_FACTORS - WT_5_luna.raw Sample Source:Wild type RAW_FILE_NAME=WT_5_luna.raw SUBJECT_SAMPLE_FACTORS - WT_6_luna.raw Sample Source:Wild type RAW_FILE_NAME=WT_6_luna.raw SUBJECT_SAMPLE_FACTORS - WT_7_luna.raw Sample Source:Wild type RAW_FILE_NAME=WT_7_luna.raw SUBJECT_SAMPLE_FACTORS - WT_8_luna.raw Sample Source:Wild type RAW_FILE_NAME=WT_8_luna.raw SUBJECT_SAMPLE_FACTORS - KO_1_luna.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_1_luna.raw SUBJECT_SAMPLE_FACTORS - KO_2_luna.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_2_luna.raw SUBJECT_SAMPLE_FACTORS - KO_3_luna.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_3_luna.raw SUBJECT_SAMPLE_FACTORS - KO_4_luna.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_4_luna.raw SUBJECT_SAMPLE_FACTORS - KO_5_luna.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_5_luna.raw SUBJECT_SAMPLE_FACTORS - KO_6_luna.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_6_luna.raw SUBJECT_SAMPLE_FACTORS - KO_7_luna.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_7_luna.raw SUBJECT_SAMPLE_FACTORS - KO_8_luna.raw Sample Source:SIRT5 knockout RAW_FILE_NAME=KO_8_luna.raw #COLLECTION CO:COLLECTION_SUMMARY Wild-type (WT) and Sirt5-/- (SIRT5 KO) mice on 129 background were maintained on CO:COLLECTION_SUMMARY a standard chow diet (Teklad Global 18% Protein Rodent Diet, ENVIGO, Cat.#2018) CO:COLLECTION_SUMMARY until they were euthanized. Metabolites extracted from eight WT and eight CO:COLLECTION_SUMMARY Sirt5-/- mouse livers were analyzed by LC/MS. Intracellular metabolites and CO:COLLECTION_SUMMARY acyl-CoA from cells were harvested as described previously using 80% CO:COLLECTION_SUMMARY methanol/water as solvent (PMID: 24410464, PMID: 24894601, PMID: 25795660) and CO:COLLECTION_SUMMARY dry pellets were stored in -80 °C freezer until ready for LC-MS analysis. For CO:COLLECTION_SUMMARY acyl-CoA and 5-methyltetrahydrofolate analysis, dry pellets were reconstituted CO:COLLECTION_SUMMARY into 30 μL of sample solvent (water containing 50 mM ammonium acetate), and 8 CO:COLLECTION_SUMMARY μL was injected into the LC-MS. For non-acyl-CoA polar metabolite analysis, CO:COLLECTION_SUMMARY pellets were reconstituted into 30 μL of sample solvent CO:COLLECTION_SUMMARY (water:methanol:acetonitrile, 2:1:1, v/v), and 3 μL was injected into the CO:COLLECTION_SUMMARY LC-MS. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY Eight Wild-type (WT) and eight Sirt5-/- (SIRT5 KO) mice on 129 background were TR:TREATMENT_SUMMARY maintained on a standard chow diet (Teklad Global 18% Protein Rodent Diet, TR:TREATMENT_SUMMARY ENVIGO, Cat.#2018) until they were euthanized. Mice had free access to food and TR:TREATMENT_SUMMARY water. Mouse body weight and food intake were monitored weekly. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Intracellular metabolites and acyl-CoA from mouse liver tissues were harvested SP:SAMPLEPREP_SUMMARY as described previously using 80% methanol/water as solvent (PMID: 24410464, SP:SAMPLEPREP_SUMMARY PMID: 24894601, PMID: 25795660) and dry pellets were stored in -80 °C freezer SP:SAMPLEPREP_SUMMARY until ready for LC-MS analysis. For acyl-CoA and 5-methyltetrahydrofolate SP:SAMPLEPREP_SUMMARY analysis, dry pellets were reconstituted into 30 μL of sample solvent (water SP:SAMPLEPREP_SUMMARY containing 50 mM ammonium acetate), and 8 μL was injected into the LC-MS. For SP:SAMPLEPREP_SUMMARY non-acyl-CoA polar metabolite analysis, pellets were reconstituted into 30 μL SP:SAMPLEPREP_SUMMARY of sample solvent (water:methanol:acetonitrile, 2:1:1, v/v), and 3 μL was SP:SAMPLEPREP_SUMMARY injected into the LC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY RPLC is for acyl-CoA and folate analysis. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Phenomenex Luna C18 (100 × 2.0 mm i.d., 3 μm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS It was operated in the full-scan mode with a scan range of 150-1000 and the MS:MS_COMMENTS resolution set at 70 000 (at m/z 200).LC-MS data were analyzed using Sieve 2.0 MS:MS_COMMENTS (Thermo Scientific), and the integrated area under metabolite peak was used to MS:MS_COMMENTS compare the relative abundance of each metabolite in different samples in the MS:MS_COMMENTS same batch. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ion counts MS_METABOLITE_DATA_START Samples WT_1_luna.raw WT_2_luna.raw WT_3_luna.raw WT_4_luna.raw WT_5_luna.raw WT_6_luna.raw WT_7_luna.raw WT_8_luna.raw KO_1_luna.raw KO_2_luna.raw KO_3_luna.raw KO_4_luna.raw KO_5_luna.raw KO_6_luna.raw KO_7_luna.raw KO_8_luna.raw Factors Sample Source:Wild type Sample Source:Wild type Sample Source:Wild type Sample Source:Wild type Sample Source:Wild type Sample Source:Wild type Sample Source:Wild type Sample Source:Wild type Sample Source:SIRT5 knockout Sample Source:SIRT5 knockout Sample Source:SIRT5 knockout Sample Source:SIRT5 knockout Sample Source:SIRT5 knockout Sample Source:SIRT5 knockout Sample Source:SIRT5 knockout Sample Source:SIRT5 knockout Acetyl-CoA 60110.88995 87972.99815 110622.0071 37399.20485 75816.21825 78570.0305 156827.3572 115046.2322 104438.7855 130904.9864 123934.5468 92897.0235 67066.5548 251610.2753 219695.2732 102788.2088 Propanoyl-CoA 251192.5407 283657.4577 253898.3242 101890.3948 133475.949 154573.6164 292786.3928 258224.2054 200020.6025 188000.6967 264500.5872 242075.2135 183436.3836 145768.7216 85540.8074 207760.3752 Isobutyryl-CoA/Butyryl-CoA 11941.9782 37821.66515 43479.0201 20377.35695 34550.6236 29185.351 42086.1707 29323.8693 50594.36105 43762.79465 58589.78335 55996.721 49055.6859 92704.46725 86392.8519 99891.3808 Isovaleryl-CoA 33222.75845 54679.67005 44423.5976 39923.7524 41694.1866 36181.04285 42937.72975 25542.5475 38585.7297 42479.1005 40560.83205 43673.65815 27393.3082 46809.3263 46635.5004 67010.5233 Malonyl-CoA 2528.29315 3855.25895 4187.3277 1933.6341 2560.2491 2548.18855 2927.1663 2285.0596 2666.6056 989.93955 3630.84185 1056.2954 2810.6483 1174.13875 2039.4582 2908.1493 Succinyl-CoA 31740.4901 55764.61935 76203.88735 23176.69425 29296.37395 24085.9651 61805.0599 46364.92425 53245.54955 35899.35665 85962.7104 56693.14985 60097.67395 50155.34165 26213.9267 42139.2575 3-Hydroxyisovaleryl-CoA 117876.3706 98103.7607 63191.7958 24590.3491 45880.3207 31299.70505 44704.98895 10304.00595 73189.79505 19482.3833 36948.4766 34365.8971 26709.3477 17816.378 17799.3722 20351.6531 3-Hydroxyisobutyryl-CoA /3-Hydroxybutanoyl-CoA 18285.51715 18131.53285 19326.9032 10041.6199 9916.16645 9959.8095 15933.85745 9946.73015 21614.9482 18715.3517 16633.98025 13312.7388 12756.83335 24828.57025 10391.93845 11054.8804 Hexanoyl-CoA 3561.0412 8197.4813 14828.47585 3229.6589 7071.01285 7215.457 9311.64455 5278.8773 12793.9914 10674.4332 16103.1197 11249.9561 11053.43775 17611.8319 15641.63225 16511.1291 3-Oxo-methylisocapryloyl-CoA 11780.84755 22876.63855 20704.63355 16323.68395 10821.3739 6279.5153 16777.2449 6000.6913 7629.7073 10659.2073 15341.5077 10629.09675 6959.9283 10945.1816 8809.1672 8670.42605 5-Methyltetrahydrofolate 40721.24345 22611.43605 39310.49365 27816.7478 22547.6069 21778.4088 18314.1022 18251.86415 29511.5549 18725.8126 30533.68945 20498.54695 19657.90125 21088.06265 11793.45055 23208.9292 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Chemical formula Acetyl-CoA C23H38N7O17P3S Propanoyl-CoA C24H40N7O17P3S Isobutyryl-CoA/Butyryl-CoA C25H42N7O17P3S Isovaleryl-CoA C26H44N7O17P3S Malonyl-CoA C24H38N7O19P3S Succinyl-CoA C25H40N7O19P3S 3-Hydroxyisovaleryl-CoA C26H44N7O18P3S 3-Hydroxyisobutyryl-CoA /3-Hydroxybutanoyl-CoA C25H42N7O18P3S Hexanoyl-CoA C27H46N7O17P3S 3-Oxo-methylisocapryloyl-CoA C30H50N7O18P3S 5-Methyltetrahydrofolate C20H25N7O6 METABOLITES_END #END