#METABOLOMICS WORKBENCH chrisbarlow_20200403_172959 DATATRACK_ID:1981 STUDY_ID:ST001348 ANALYSIS_ID:AN002242
VERSION                          	1
CREATED_ON                       	02-08-2024
#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
PR:DOI                           	http://dx.doi.org/10.21228/M8568J
#STUDY
ST:STUDY_TITLE                   	Metabolomic changes in mouse liver on a threonine restricted diet (part-III)
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). The data in this study corresponds to liver from C57Bl/6JMarp mice
ST:STUDY_SUMMARY                 	subject to a 3wk treatment with diets either containing 18% digestible energy
ST:STUDY_SUMMARY                 	from amino acids with either a normal distribution of amino acids or with
ST:STUDY_SUMMARY                 	restricted amounts of threonine. This followed prior treatment with
ST:STUDY_SUMMARY                 	adeno-associated viruses to transduce the liver to express yeast threonine
ST:STUDY_SUMMARY                 	biosynthetic enzymes (AAV-yTHR1+THR4) or a negative control (AAV-GFP). n= 6
ST:STUDY_SUMMARY                 	individual mice per 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:SUBMIT_DATE                   	2020-04-03
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:GENOTYPE_STRAIN               	C57Bl/6JMarp
SU:AGE_OR_AGE_RANGE              	7 to 12 weeks
SU:GENDER                        	Male
SU:ANIMAL_ANIMAL_SUPPLIER        	Monash University Animal Research Platform, Clayton, AUS
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	AR401_control_AA_GFP_1pt1	Diet:Control diet SF17-177 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR401_control_AA_GFP_1pt1_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR402_control_AA_GFP_1pt2	Diet:Control diet SF17-177 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR402_control_AA_GFP_1pt2_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR403_control_AA_GFP_1pt3	Diet:Control diet SF17-177 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR403_control_AA_GFP_1pt3_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR404_control_AA_GFP_2pt1	Diet:Control diet SF17-177 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR404_control_AA_GFP_2pt1_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR405_control_AA_GFP_2pt2	Diet:Control diet SF17-177 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR405_control_AA_GFP_2pt2_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR406_control_AA_GFP_2pt3	Diet:Control diet SF17-177 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR406_control_AA_GFP_2pt3_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR407_control_AA_THR1plus4_3pt1	Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR407_control_AA_THR1plus4_3pt1_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR408_control_AA_THR1plus4_3pt2	Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR408_control_AA_THR1plus4_3pt2_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR409_control_AA_THR1plus4_3pt3	Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR409_control_AA_THR1plus4_3pt3_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR410_control_AA_THR1plus4_4pt1	Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR410_control_AA_THR1plus4_4pt1_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR411_control_AA_THR1plus4_4pt2	Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR411_control_AA_THR1plus4_4pt2_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR412_control_AA_THR1plus4_4pt3	Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR412_control_AA_THR1plus4_4pt3_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR413_Low_T_GFP_5pt1	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR413_Low_T_GFP_5pt1_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR414_Low_T_GFP_5pt2	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR414_Low_T_GFP_5pt2_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR415_Low_T_GFP_5pt3	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR415_Low_T_GFP_5pt3_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR416_Low_T_GFP_6pt1	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR416_Low_T_GFP_6pt1_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR417_Low_T_GFP_6pt2	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR417_Low_T_GFP_6pt2_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR418_Low_T_GFP_6pt3	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP	RAW_FILE_NAME=C120181105_AR418_Low_T_GFP_6pt3_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR419_Low_T_THR1plus4_7pt1	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR419_Low_T_THR1plus4_7pt1_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR420_Low_T_THR1plus4_7pt2	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR420_Low_T_THR1plus4_7pt2_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR421_Low_T_THR1plus4_7pt3	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR421_Low_T_THR1plus4_7pt3_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR422_Low_T_THR1plus4_8pt1	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR422_Low_T_THR1plus4_8pt1_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR423_Low_T_THR1plus4_8pt2	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR423_Low_T_THR1plus4_8pt2_1.raw
SUBJECT_SAMPLE_FACTORS           	-	AR424_Low_T_THR1plus4_8pt3	Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4	RAW_FILE_NAME=C120181105_AR424_Low_T_THR1plus4_8pt3_1.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Mice were 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 (8720610, Barastoc, AUS).
TR:TREATMENT_SUMMARY             	Following acclimation, mice were administered a total of 5E+11 virus particles
TR:TREATMENT_SUMMARY             	per mouse via the tail vein. For the negative control (NC): 5E+11 GFP-AAV; and
TR:TREATMENT_SUMMARY             	for the THR1/4 overexpression studies mice were administered 2.5E+11 virus
TR:TREATMENT_SUMMARY             	particles each of yTHR1-AAV and yTHR4-AAV. One week following this time, the
TR:TREATMENT_SUMMARY             	dietary intervention was initiated. In these studies, the low threonine diet
TR:TREATMENT_SUMMARY             	(SF18-066, Specialty Feeds AUS) contained homoserine, the substrate of THR1. The
TR:TREATMENT_SUMMARY             	corresponding control diet was SF17-177 (Specialty Feeds AUS).
#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. Additionally where quantitative amino acid
SP:SAMPLEPREP_SUMMARY            	analysis was performed, a mixture of 17 stable isotope labelled amino acids
SP:SAMPLEPREP_SUMMARY            	(Gln, Asn and Trp were absent) were added at a concentration of 500 pmol of each
SP:SAMPLEPREP_SUMMARY            	amino acid per mg liver (Cambridge Isotope Laboratories PN MSK-A2-1.2).
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:INSTRUMENT_NAME               	Thermo Dionex Ultimate 3000 RS
CH:COLUMN_NAME                   	SeQuant ZIC-pHILIC (150 x 4.6mm,5um)
CH:COLUMN_TEMPERATURE            	25
CH:FLOW_GRADIENT                 	linear gradient time-%B as follows: 0 min-80%, 15 min-50%, 18 min-5%, 21 min-5%,
CH:FLOW_GRADIENT                 	24 min-80%, 32 min-80%
CH:FLOW_RATE                     	300 µL/min
CH:SOLVENT_A                     	100% water; 20 mM ammonium carbonate
CH:SOLVENT_B                     	100% water; acetonitrile
CH:CHROMATOGRAPHY_TYPE           	HILIC
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	Mass spectrometry was performed at 35 000 resolution (accuracy calibrated to
MS:MS_COMMENTS                   	<1 ppm) on a Q-Exactive Orbitrap MS (Thermo) operating in rapid switching
MS:MS_COMMENTS                   	positive (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:ION_MODE                      	UNSPECIFIED
MS:MS_RESULTS_FILE               	ST001348_AN002242_Results.txt	UNITS:Peak Area	Has RT:Yes	RT units:Minutes
#END