#METABOLOMICS WORKBENCH chrisbarlow_20200401_204809 DATATRACK_ID:1972 STUDY_ID:ST001346 ANALYSIS_ID:AN002240 PROJECT_ID:PR000917 VERSION 1 CREATED_ON April 5, 2020, 5:38 pm #PROJECT PR:PROJECT_TITLE Restriction of essential amino acids dictates the systemic metabolic response to PR:PROJECT_TITLE dietary protein dilution PR:PROJECT_SUMMARY The data provided here are in support of the publication “Restriction of PR:PROJECT_SUMMARY essential amino acids dictates the systemic metabolic response to dietary PR:PROJECT_SUMMARY protein dilution” Yann W. Yap et al. Nature Communications 2020 (accepted for PR:PROJECT_SUMMARY publication). Here we provide untargeted metabolomics LC-MS data from liver and PR:PROJECT_SUMMARY plasma from mice fed a diet in which dietary protein was restricted and PR:PROJECT_SUMMARY corresponding unrestricted controls. Specifically, part 1 and 2 correspond to PR:PROJECT_SUMMARY liver and plasma from the hepatic portal vein respectively from animals on a PR:PROJECT_SUMMARY low-protein diet following a week of diet adaptation and correspond controls PR:PROJECT_SUMMARY with n = 5 for each group. Part 3 of the data presented here corresponds to PR:PROJECT_SUMMARY liver from mice subject to a 3wk treatment with diets either containing 18% PR:PROJECT_SUMMARY digestible energy from amino acids or a diet with restricted amounts of PR:PROJECT_SUMMARY threonine and following prior treatments with adeno-associated viruses to PR:PROJECT_SUMMARY transduce the liver to express yeast threonine biosynthetic enzymes PR:PROJECT_SUMMARY (AAV-yTHR1+THR4) or a negative control (AAV-GFP). n= 6 individual mice per PR:PROJECT_SUMMARY group. PR:INSTITUTE Monash University PR:DEPARTMENT Department of Biochemistry and Molecular Biology PR:LABORATORY Nutrient Metabolism and Signalling Laboratory PR:LAST_NAME Rose PR:FIRST_NAME Adam PR:ADDRESS Monash University, Clayton Campus, 23 Innovation Walk, Clayton, Victoria, 3800, PR:ADDRESS Australia PR:EMAIL adam.rose@monash.edu PR:PHONE +61 3 99029340 #STUDY ST:STUDY_TITLE Metabolomic changes in mouse liver on a protein restricted diet ST:STUDY_SUMMARY The data provided here are in support of the publication “Restriction of ST:STUDY_SUMMARY essential amino acids dictates the systemic metabolic response to dietary ST:STUDY_SUMMARY protein dilution” Yann W. Yap et al. Nature Communications 2020 (accepted for ST:STUDY_SUMMARY publication). Here we provide untargeted metabolomics LC-MS data from liver from ST:STUDY_SUMMARY C57Bl/6NCrl mice fed a diet in which dietary protein was restricted and ST:STUDY_SUMMARY corresponding unrestricted controls. Specifically, liver from animals on a ST:STUDY_SUMMARY low-protein diet following a week of diet adaptation and correspond controls ST:STUDY_SUMMARY with n = 5 for each group. ST:INSTITUTE Monash University ST:DEPARTMENT Department of Biochemistry and Molecular Biology ST:LABORATORY Nutrient Metabolism and Signalling Laboratory ST:LAST_NAME Rose ST:FIRST_NAME Adam ST:ADDRESS Monash University, Clayton Campus, 23 Innovation Walk, Clayton, Victoria, 3800, ST:ADDRESS Australia ST:EMAIL adam.rose@monash.edu ST:PHONE +61 3 99029340 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 10 ST:NUM_MALES 10 ST:STUDY_COMMENTS Study 1 of 3 for this project #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57Bl/6NCrl SU:AGE_OR_AGE_RANGE 7 to 10 weeks SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Charles River Laboratories, DEU SU:ANIMAL_LIGHT_CYCLE 12-12h light-dark cycle #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 - AR101_1-1 Diet:Control (normal protein diet) D12450B RAW_FILE_NAME=C120180704_AR101_1-1_1.raw SUBJECT_SAMPLE_FACTORS - AR102_1-2 Diet:Control (normal protein diet) D12450B RAW_FILE_NAME=C120180704_AR102_1-2_1.raw SUBJECT_SAMPLE_FACTORS - AR103_1-3 Diet:Control (normal protein diet) D12450B RAW_FILE_NAME=C120180704_AR103_1-3_1.raw SUBJECT_SAMPLE_FACTORS - AR104_1-4 Diet:Control (normal protein diet) D12450B RAW_FILE_NAME=C120180704_AR104_1-4_1.raw SUBJECT_SAMPLE_FACTORS - AR105_1-5 Diet:Control (normal protein diet) D12450B RAW_FILE_NAME=C120180704_AR105_1-5_1.raw SUBJECT_SAMPLE_FACTORS - AR106_2-1 Diet:Reduced protein diet D10062201 RAW_FILE_NAME=C120180704_AR106_2-1_1.raw SUBJECT_SAMPLE_FACTORS - AR107_2-2 Diet:Reduced protein diet D10062201 RAW_FILE_NAME=C120180704_AR107_2-2_1.raw SUBJECT_SAMPLE_FACTORS - AR108_2-3 Diet:Reduced protein diet D10062201 RAW_FILE_NAME=C120180704_AR108_2-3_1.raw SUBJECT_SAMPLE_FACTORS - AR109_2-4 Diet:Reduced protein diet D10062201 RAW_FILE_NAME=C120180704_AR109_2-4_1.raw SUBJECT_SAMPLE_FACTORS - AR110_2-5 Diet:Reduced protein diet D10062201 RAW_FILE_NAME=C120180704_AR110_2-5_1.raw #COLLECTION CO:COLLECTION_SUMMARY Mice were fasted overnight and then re-fed for 5 to 6 hours before being CO:COLLECTION_SUMMARY humanely euthanised for tissue collection. CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Male mice aged 7 weeks upon arrival, were acclimated to the local housing TR:TREATMENT_SUMMARY facility (12-12h light-dark cycle, 22-24°C) for one week prior to TR:TREATMENT_SUMMARY experimentation and were fed standard rodent chow (3437, Provimi Kliba, DEU). TR:TREATMENT_SUMMARY Following acclimation, mice were placed on either a normal protein diet TR:TREATMENT_SUMMARY (D12450B, Research Diets, New Jersey, USA) or a reduced protein diet (D10062201, TR:TREATMENT_SUMMARY Research Diets, New Jersey, USA) for 3 weeks prior to being humanely euthanised TR:TREATMENT_SUMMARY for tissue collection. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Livers were cryogenically pulverized (cryopulverization) using a 12-well SP:SAMPLEPREP_SUMMARY biopulverizer (BioSpec Products, OK USA Part number 59012MS) according to the SP:SAMPLEPREP_SUMMARY manufacturer’s instructions. The frozen tissue powder was then weighed and SP:SAMPLEPREP_SUMMARY extracted in 20 µL of extraction solvent (0°C) per mg of tissue. The mixture SP:SAMPLEPREP_SUMMARY was then briefly vortexed before sonication in an ice-water bath for 10 minutes SP:SAMPLEPREP_SUMMARY followed by centrifugation (20,000 rcf, 4℃, 10 minutes). The supernatant was SP:SAMPLEPREP_SUMMARY then transferred to a mass spectrometry vial for LC-MS analysis. The extraction SP:SAMPLEPREP_SUMMARY solvent consisted of 2:6:1 CHCl3:MeOH:H2O v/v/v with 2 µM CHAPS, CAPS, PIPES SP:SAMPLEPREP_SUMMARY and TRIS as internal standards. SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatography utilized a ZIC-pHILIC column (column temperature 25 °C) with a CH:CHROMATOGRAPHY_SUMMARY gradient elution of 20 mM ammonium carbonate (A) and acetonitrile (B) (linear CH:CHROMATOGRAPHY_SUMMARY gradient time-%B as follows: 0 min-80%, 15 min-50%, 18 min-5%, 21 min-5%, 24 CH:CHROMATOGRAPHY_SUMMARY min-80%, 32 min-80%) on a Dionex RSLC3000 UHPLC (Thermo). The flow rate was CH:CHROMATOGRAPHY_SUMMARY maintained at 300 μL/min. Samples were kept at 4 °C in the autosampler and 10 CH:CHROMATOGRAPHY_SUMMARY μL injected for analysis. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME SeQuant ZIC- pHILIC (150 x 4.6 mm, 5uM) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Mass spectrometry was performed at 35 000 resolution (accuracy calibrated to <1 MS:MS_COMMENTS ppm) on a Q-Exactive Orbitrap MS (Thermo) operating in rapid switching positive MS:MS_COMMENTS (4 kV) and negative (−3.5 kV) mode electrospray ionization (capillary MS:MS_COMMENTS temperature 300 °C; sheath gas 50; Aux gas 20; sweep gas 2; probe temp 120 MS:MS_COMMENTS °C). MS:MS_RESULTS_FILE ST001346_AN002240_Results.txt UNITS:Peak Area Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END