#METABOLOMICS WORKBENCH zhaom_20220406_224236 DATATRACK_ID:3181 STUDY_ID:ST002127 ANALYSIS_ID:AN003480 VERSION 1 CREATED_ON 05-31-2022 #PROJECT PR:PROJECT_TITLE Amino acid catabolism in hematopoietic cells PR:PROJECT_SUMMARY Hematopoietic stem cells (HSCs) adapt their metabolism to maintenance and PR:PROJECT_SUMMARY proliferation, but the mechanism remains incompletely understood. Here, we have PR:PROJECT_SUMMARY investigated the total levels, uptake and catabolism of amino acid in PR:PROJECT_SUMMARY hematopoietic stem cells, hemopoietic progenitors, and differentiated PR:PROJECT_SUMMARY hematopoietic cells. We have also studied the catabolism of amino acid in PR:PROJECT_SUMMARY hematopoietic stem cells under different conditions, such as homeostasis and PR:PROJECT_SUMMARY proliferation and with drug treatment. Moreover, glycolytic metabolite, NAD+ PR:PROJECT_SUMMARY precursor nicotinamide riboside (NR), accelerated AA catabolism to activate GCN2 PR:PROJECT_SUMMARY and sustain long-term function of HSCs. Overall, our study uncovers the direct PR:PROJECT_SUMMARY links between metabolic alterations and translation control in HSCs during PR:PROJECT_SUMMARY homeostasis and proliferation. PR:INSTITUTE Sun Yat-sen University PR:LAST_NAME Zhao PR:FIRST_NAME Meng PR:ADDRESS Zhongshan 2nd Road PR:EMAIL zhaom38@mail.sysu.edu.cn PR:PHONE 18138799889 PR:DOI http://dx.doi.org/10.21228/M8GD8D #STUDY ST:STUDY_TITLE Amino acids and TCA substrates in hematopoietic cells (Part4) ST:STUDY_SUMMARY This study uses [13C,15N] labeled amino acids to study the amino acid ST:STUDY_SUMMARY consumption and their catabolism into tricarboxylic acid cycle substrates in ST:STUDY_SUMMARY hematopoietic stem cells, hemopoietic progenitors, and differentiated ST:STUDY_SUMMARY hematopoietic cells under different conditions, such as homeostasis and ST:STUDY_SUMMARY proliferation and with drug treatment. ST:INSTITUTE Sun Yat-sen University ST:LAST_NAME Zhao ST:FIRST_NAME Meng ST:ADDRESS Zhongshan 2nd Road ST:EMAIL zhaom38@mail.sysu.edu.cn ST:PHONE 18138799889 ST:SUBMIT_DATE 2022-04-06 #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 - HSC_sot_ND_1 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) RAW_FILE_NAME=hsc-sot-nd-1.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_ND_2 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) RAW_FILE_NAME=hsc-sot-nd-2.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_ND_3 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) RAW_FILE_NAME=hsc-sot-nd-3.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_ND_0h_1 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nd-0h-1.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_ND_0h_2 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nd-0h-2.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_ND_0h_3 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nd-0h-3.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_ND_12h_1 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nd-12h-1.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_ND_12h_2 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nd-12h-2.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_ND_12h_3 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nd-12h-3.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_ND_6h_1 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nd-6h-1.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_ND_6h_2 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nd-6h-2.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_ND_6h_3 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nd-6h-3.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_1 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) RAW_FILE_NAME=hsc-sot-nr-1.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_2 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) RAW_FILE_NAME=hsc-sot-nr-2.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_3 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) RAW_FILE_NAME=hsc-sot-nr-3.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_0h_1 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nr-0h-1.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_0h_2 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nr-0h-2.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_0h_3 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nr-0h-3.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_12h_1 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nr-12h-1.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_12h_2 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nr-12h-2.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_12h_3 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nr-12h-3.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_6h_1 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nr-6h-1.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_6h_2 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nr-6h-2.cdf SUBJECT_SAMPLE_FACTORS - HSC_sot_NR_6h_3 Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). RAW_FILE_NAME=hsc-sot-nr-6h-3.cdf #COLLECTION CO:COLLECTION_SUMMARY 5×10^5 indicated cells were incubated with 100 µM stable [13C,15N] amino acids CO:COLLECTION_SUMMARY (MSK-A2-US-1.2, Cambridge Isotope Laboratories) for indicated time and CO:COLLECTION_SUMMARY centrifuged at 500 g for 5 min at 4 °C. The pelleted cells were extracted in CO:COLLECTION_SUMMARY 500 µl ice-cold Acetonitrile: Isopropyl Alcohol: water (3:3:2 v/v/v) and CO:COLLECTION_SUMMARY aliquoted as three technical replicates. The extracts were vortexed for 5 min at CO:COLLECTION_SUMMARY 4 °C and centrifuged at 14,000 g for 2 min at 4 °C. The supernatants were CO:COLLECTION_SUMMARY dried by vacuum spin for subsequent derivatization and stored at -20 ℃. CO:SAMPLE_TYPE Bone marrow #TREATMENT TR:TREATMENT_SUMMARY Germ-free C57BL/6J mice were intraperitoneally injected with 10 mg/kg 5FU TR:TREATMENT_SUMMARY (F6627-5G, Sigma-Aldrich) for 14 days, or fed with vehicle or NR (400 mg/kg per TR:TREATMENT_SUMMARY day) (1341-23-7, ziyi-reagent) for consecutive 8 weeks (long-term) or 1 week TR:TREATMENT_SUMMARY (short-term). Hematopoietic cells were stained and sorted from treated mice. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To get the corresponding derivatives, the dried aliquots were incubated with 20 SP:SAMPLEPREP_SUMMARY µL 2% (w/v) methoxyamine hydrochloride (226904, Sigma-Aldrich) in pyridine for SP:SAMPLEPREP_SUMMARY 60 min at 37 °C, and silylated by 30 µL of SP:SAMPLEPREP_SUMMARY N-Methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide with 1% SP:SAMPLEPREP_SUMMARY tert-Butyldimethylchlorosilane (TBDMS, 18162-48-6, Regis Technologies) for 30 SP:SAMPLEPREP_SUMMARY min at 45 °C. The corresponding derivatives were analyzed by GC-MS using the SP:SAMPLEPREP_SUMMARY Trace 1310 gas chromatograph (Thermo Fisher) with the DB-35ms column (Agilent SP:SAMPLEPREP_SUMMARY Technologies) connected to the Q ExactiveTM GC OrbitrapTM GC-MS/MS system SP:SAMPLEPREP_SUMMARY (Thermo Fisher). #CHROMATOGRAPHY CH:INSTRUMENT_NAME TRACE 1310 CH:COLUMN_NAME DB-35ms CH:CHROMATOGRAPHY_TYPE GC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE EI MS:MS_COMMENTS GC-MS data was acquired on Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) MS:MS_COMMENTS coupled with Gas Chromatograph system TRACE 1310 (Thermo Fisher). MS:MS_COMMENTS Chromatographic separation was performed on a DB-35ms column module (30 m length MS:MS_COMMENTS x 0.25 mm internal diameter, Agilent Technologies). The column temperature was MS:MS_COMMENTS programmed with an initial temperature of 50 °C for 2 min, then ramped at MS:MS_COMMENTS 10°C/min to 325 °C, and maintained for 5 min. The mass range was set as 50-600 MS:MS_COMMENTS m/z, and the resolution was 60,000. The ion source temperature was 300°C with MS:MS_COMMENTS the transfer line temperature of 250°C, and the electron energy was 70 eV with MS:MS_COMMENTS EI source. MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples HSC_sot_ND_1 HSC_sot_ND_2 HSC_sot_ND_3 HSC_sot_ND_0h_1 HSC_sot_ND_0h_2 HSC_sot_ND_0h_3 HSC_sot_ND_12h_1 HSC_sot_ND_12h_2 HSC_sot_ND_12h_3 HSC_sot_ND_6h_1 HSC_sot_ND_6h_2 HSC_sot_ND_6h_3 HSC_sot_NR_1 HSC_sot_NR_2 HSC_sot_NR_3 HSC_sot_NR_0h_1 HSC_sot_NR_0h_2 HSC_sot_NR_0h_3 HSC_sot_NR_12h_1 HSC_sot_NR_12h_2 HSC_sot_NR_12h_3 HSC_sot_NR_6h_1 HSC_sot_NR_6h_2 HSC_sot_NR_6h_3 Factors Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:Ctrl, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 1 h | Batch:1e | Note:[13C, 15N]AAs were used for AA uptake (FigS6.J) Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 0 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 12 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). Source_Name[gating]:Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) | Treatment:NR 1 week, [13C, 15N] AAs were incorporated for 24 h and subsequently washed out for 6 h | Batch:1e | Note:[13C, 15N] AA levels and [13C]TCA substrate levels in different time point were calculated for AA catabolism (Fig5.L). [13C_ 15N]Alanine_ 2TBDMS 331000.0000 317000.0000 310000.0000 3310000.0000 3170000.0000 3100000.0000 875000.0000 921000.0000 830000.0000 1600000.0000 1410000.0000 1360000.0000 332000.0000 299000.0000 312000.0000 3320000.0000 2990000.0000 3120000.0000 753000.0000 727000.0000 691000.0000 1330000.0000 1240000.0000 1290000.0000 [13C_ 15N]Aspartic acid_ 3TBDMS 82500.0000 83500.0000 74300.0000 825000.0000 835000.0000 743000.0000 365000.0000 321000.0000 333000.0000 599000.0000 435000.0000 509000.0000 84000.0000 76900.0000 79700.0000 840000.0000 769000.0000 797000.0000 313000.0000 303000.0000 296000.0000 543000.0000 486000.0000 423000.0000 [13C_ 15N]Glutamic acid_ 3TBDMS 61300.0000 63800.0000 60700.0000 613000.0000 638000.0000 607000.0000 235000.0000 244000.0000 207000.0000 395000.0000 407000.0000 385000.0000 66700.0000 57100.0000 59100.0000 667000.0000 571000.0000 591000.0000 184000.0000 182000.0000 154000.0000 310000.0000 279000.0000 288000.0000 [13C_ 15N]Glycine_ 2TBDMS 51100.0000 63400.0000 50800.0000 6350000.0000 5500000.0000 5380000.0000 3040000.0000 3010000.0000 2070000.0000 3840000.0000 4870000.0000 4100000.0000 58700.0000 59500.0000 57400.0000 6710000.0000 5780000.0000 5350000.0000 3130000.0000 2930000.0000 3380000.0000 3880000.0000 4920000.0000 4900000.0000 [13C_ 15N]Histidine_ 3TBDMS 64800.0000 56500.0000 57700.0000 648000.0000 565000.0000 577000.0000 173000.0000 127000.0000 208000.0000 314000.0000 313000.0000 354000.0000 54600.0000 54400.0000 49500.0000 546000.0000 544000.0000 495000.0000 102000.0000 136000.0000 138000.0000 221000.0000 314000.0000 242000.0000 [13C_ 15N]Isoleucine_ 2TBDMS 328000.0000 306000.0000 281000.0000 3280000.0000 3060000.0000 2810000.0000 1400000.0000 1430000.0000 1100000.0000 2150000.0000 2250000.0000 2140000.0000 296000.0000 384000.0000 357000.0000 2960000.0000 3840000.0000 3570000.0000 1370000.0000 1040000.0000 995000.0000 2270000.0000 2300000.0000 2130000.0000 [13C_ 15N]Leucine_ 2TBDMS 369000.0000 353000.0000 318000.0000 3690000.0000 3530000.0000 3180000.0000 1040000.0000 984000.0000 1080000.0000 2120000.0000 2240000.0000 2140000.0000 302000.0000 353000.0000 321000.0000 3020000.0000 3530000.0000 3210000.0000 782000.0000 810000.0000 741000.0000 1460000.0000 1580000.0000 1900000.0000 [13C_ 15N]Lysine_ 3TBDMS 148000.0000 139000.0000 133000.0000 1480000.0000 1390000.0000 1330000.0000 594000.0000 551000.0000 585000.0000 813000.0000 786000.0000 796000.0000 134000.0000 142000.0000 166000.0000 1340000.0000 1420000.0000 1660000.0000 466000.0000 441000.0000 464000.0000 766000.0000 780000.0000 837000.0000 [13C_ 15N]Methionine_ 2TBDMS 197000.0000 206000.0000 182000.0000 1970000.0000 2060000.0000 1820000.0000 740000.0000 680000.0000 544000.0000 1200000.0000 1180000.0000 927000.0000 200000.0000 220000.0000 173000.0000 2000000.0000 2200000.0000 1730000.0000 501000.0000 575000.0000 607000.0000 1010000.0000 1270000.0000 1040000.0000 [13C_ 15N]Phenylalanine_ 2TBDMS 289000.0000 297000.0000 243000.0000 2890000.0000 2970000.0000 2430000.0000 1240000.0000 1380000.0000 1130000.0000 1410000.0000 1520000.0000 1440000.0000 246000.0000 276000.0000 222000.0000 2460000.0000 2760000.0000 2220000.0000 1000000.0000 929000.0000 926000.0000 1360000.0000 1240000.0000 1360000.0000 [13C_ 15N]Proline_ 2TBDMS 184000.0000 168000.0000 174000.0000 1840000.0000 1680000.0000 1740000.0000 450000.0000 528000.0000 423000.0000 866000.0000 829000.0000 857000.0000 176000.0000 161000.0000 160000.0000 1760000.0000 1610000.0000 1600000.0000 319000.0000 275000.0000 325000.0000 750000.0000 701000.0000 731000.0000 [13C_ 15N]Serine_ 3TBDMS 321000.0000 324000.0000 282000.0000 3210000.0000 3240000.0000 2820000.0000 964000.0000 851000.0000 886000.0000 2120000.0000 2190000.0000 1860000.0000 347000.0000 311000.0000 356000.0000 3470000.0000 3110000.0000 3560000.0000 712000.0000 793000.0000 822000.0000 1940000.0000 1880000.0000 1650000.0000 [13C_ 15N]Threonine_ 3TBDMS 86000.0000 79700.0000 77200.0000 860000.0000 797000.0000 772000.0000 342000.0000 313000.0000 322000.0000 510000.0000 508000.0000 413000.0000 61200.0000 67200.0000 85200.0000 612000.0000 672000.0000 852000.0000 286000.0000 244000.0000 232000.0000 362000.0000 350000.0000 368000.0000 [13C_ 15N]Tyrosine_ 3TBDMS 798000.0000 774000.0000 724000.0000 7980000.0000 7740000.0000 7240000.0000 2250000.0000 2370000.0000 2940000.0000 4140000.0000 4380000.0000 3180000.0000 730000.0000 698000.0000 740000.0000 7300000.0000 6980000.0000 7400000.0000 2210000.0000 1830000.0000 1850000.0000 3120000.0000 3970000.0000 3320000.0000 [13C_ 15N]Valine_ 2TBDMS 805000.0000 762000.0000 793000.0000 8050000.0000 7620000.0000 7930000.0000 3210000.0000 2980000.0000 2570000.0000 5270000.0000 4440000.0000 4280000.0000 822000.0000 769000.0000 787000.0000 8220000.0000 7690000.0000 7870000.0000 1790000.0000 2280000.0000 2610000.0000 4020000.0000 4230000.0000 4080000.0000 [13C]Alpha_Ketoglutarate 2160000.0000 1880000.0000 2000000.0000 34000.0000 37400.0000 41200.0000 26100.0000 28500.0000 30100.0000 23500.0000 20500.0000 21700.0000 50900.0000 51500.0000 50900.0000 33400.0000 32100.0000 35000.0000 [13C]Citrate 45400.0000 42100.0000 39700.0000 81800.0000 80700.0000 86800.0000 70200.0000 69300.0000 69800.0000 48900.0000 55500.0000 46700.0000 107000.0000 90600.0000 95600.0000 82200.0000 84200.0000 82400.0000 [13C]Fumatate 173000.0000 176000.0000 142000.0000 343000.0000 315000.0000 342000.0000 244000.0000 249000.0000 275000.0000 159000.0000 138000.0000 138000.0000 364000.0000 354000.0000 367000.0000 293000.0000 276000.0000 285000.0000 [13C]Malate 84900.0000 90700.0000 104000.0000 142000.0000 162000.0000 145000.0000 116000.0000 136000.0000 133000.0000 108000.0000 137000.0000 119000.0000 164000.0000 178000.0000 184000.0000 141000.0000 150000.0000 156000.0000 [13C]Succinate 128000.0000 135000.0000 113000.0000 213000.0000 235000.0000 231000.0000 192000.0000 160000.0000 185000.0000 108000.0000 111000.0000 123000.0000 247000.0000 246000.0000 267000.0000 209000.0000 192000.0000 206000.0000 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 [13C, 15N]Alanine, 2TBDMS 15.06 [13C, 15N]Aspartic acid, 3TBDMS 21.49 [13C, 15N]Glutamic acid, 3TBDMS 22.54 [13C, 15N]Glycine, 2TBDMS 15.39 [13C, 15N]Histidine, 3TBDMS 25.11 [13C, 15N]Isoleucine, 2TBDMS 17.26 [13C, 15N]Leucine, 2TBDMS 16.91 [13C, 15N]Lysine, 3TBDMS 23.48 [13C, 15N]Methionine, 2TBDMS 19.74 [13C, 15N]Phenylalanine, 2TBDMS 20.92 [13C, 15N]Proline, 2TBDMS 20.49 [13C, 15N]Serine, 3TBDMS 19.94 [13C, 15N]Threonine, 3TBDMS 20.22 [13C, 15N]Tyrosine, 3TBDMS 25.51 [13C, 15N]Valine, 2TBDMS 16.46 [13C]Alpha_Ketoglutarate 21.03 [13C]Citrate 26.51 [13C]Fumatate 18.77 [13C]Malate 22.05 [13C]Succinate 18.37 METABOLITES_END #END