#METABOLOMICS WORKBENCH dougguzior_20250326_111206 DATATRACK_ID:5791 STUDY_ID:ST004085 ANALYSIS_ID:AN006769 PROJECT_ID:PR002564
VERSION             	1
CREATED_ON             	April 4, 2025, 5:30 pm
#PROJECT
PR:PROJECT_TITLE                 	Dietary methionine restriction in high-fat diet-fed male mice
PR:PROJECT_SUMMARY               	25-week-old male mice (n=31) with diet-induced obesity were divided into four
PR:PROJECT_SUMMARY               	groups. The first group (n=8) was fed a high-fat diet (HFD, 60% kcal fat) for 15
PR:PROJECT_SUMMARY               	weeks. The second, third, and fourth groups were fed a modified high-fat diet
PR:PROJECT_SUMMARY               	(HFD-MetR, 60% fat/0.12% methionine) for 3 days (MetR3, n=8), 5 days (MetR5,
PR:PROJECT_SUMMARY               	n=8), or 10 days (MetR10, n=7), respectively. The HFD group (60% fat/0.86%
PR:PROJECT_SUMMARY               	methionine) served as the control. Mice were fasted for 4 hours prior to sample
PR:PROJECT_SUMMARY               	collection. The goal of this study was to investigate the effect of methionine
PR:PROJECT_SUMMARY               	restriction on plasma, brown adipose, and liver metabolomes.
PR:INSTITUTE                     	Panome Bio
PR:LAST_NAME                     	Guzior
PR:FIRST_NAME                    	Douglas
PR:ADDRESS                       	4340 Duncan Ave, St. Louis, MO 63110
PR:EMAIL                         	douglas.guzior@panomebio.com
PR:PHONE                         	(314) 632-6588
#STUDY
ST:STUDY_TITLE                   	Dietary methionine restriction in high-fat diet-fed male mice, plasma
ST:STUDY_TITLE                   	metabolomics
ST:STUDY_SUMMARY                 	25-week-old male mice (n=31) with diet-induced obesity were divided into four
ST:STUDY_SUMMARY                 	groups. The first group (n=8) was fed a high-fat diet (HFD, 60% kcal fat) for 15
ST:STUDY_SUMMARY                 	weeks. The second, third, and fourth groups were fed a modified high-fat diet
ST:STUDY_SUMMARY                 	(HFD-MetR, 60% fat/0.12% methionine) for 3 days (MetR3, n=8), 5 days (MetR5,
ST:STUDY_SUMMARY                 	n=8), or 10 days (MetR10, n=7), respectively. The HFD group (60% fat/0.86%
ST:STUDY_SUMMARY                 	methionine) served as the control. Mice were fasted for 4 hours prior to sample
ST:STUDY_SUMMARY                 	collection. The goal of this study was to investigate the effect of methionine
ST:STUDY_SUMMARY                 	restriction on brown adipose tissues.
ST:INSTITUTE                     	Panome Bio
ST:LAST_NAME                     	Guzior
ST:FIRST_NAME                    	Douglas
ST:ADDRESS                       	4340 Duncan Ave, St. Louis, MO 63110
ST:EMAIL                         	douglas.guzior@panomebio.com
ST:PHONE                         	(314) 632-6588
ST:NUM_GROUPS                    	4
ST:TOTAL_SUBJECTS                	31
ST:NUM_MALES                     	31
ST:STUDY_COMMENTS                	Part 3 of 3
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:GENOTYPE_STRAIN               	C57BL/6J
SU:AGE_OR_AGE_RANGE              	25 wk
SU:WEIGHT_OR_WEIGHT_RANGE        	>45g bodyweight at 25 w/o
SU:ANIMAL_LIGHT_CYCLE            	12-hour light/12-hour dark photoperiod (0700 – 1900)
SU:ANIMAL_FEED                   	Research Diets D12492; Research Diets A14032002; Research Diets A14032001
SU:ANIMAL_INCLUSION_CRITERIA     	>45g bodyweight at 25 w/o
#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           	-	CF-5	group:Control | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-001_009.mzML
SUBJECT_SAMPLE_FACTORS           	-	CF-6	group:Control | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-002_016.mzML
SUBJECT_SAMPLE_FACTORS           	-	CF-7	group:Control | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-003_007.mzML
SUBJECT_SAMPLE_FACTORS           	-	CF-8	group:Control | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-004_010.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR3-9	group:MetR3 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-005_017.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR3-13	group:MetR3 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-006_018.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR3-14	group:MetR3 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-007_012.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR3-15	group:MetR3 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-008_006.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR3-16	group:MetR3 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-009_014.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR5-21	group:MetR5 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-010_005.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR5-22	group:MetR5 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-011_020.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR5-23	group:MetR5 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-012_008.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR5-24	group:MetR5 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-013_019.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR10-29	group:MetR10 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-014_011.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR10-30	group:MetR10 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-015_013.mzML
SUBJECT_SAMPLE_FACTORS           	-	MR10-31	group:MetR10 | Sample source:Plasma	RAW_FILE_NAME(filename)=060-0145-016_015.mzML
SUBJECT_SAMPLE_FACTORS           	-	blank1	group:blank | Sample source:blank	RAW_FILE_NAME(filename)=060-0145-Blank_3_027.mzML
SUBJECT_SAMPLE_FACTORS           	-	qc1	group:qc | Sample source:pooled_qc	RAW_FILE_NAME(filename)=060-0145-QC_1_003.mzML
SUBJECT_SAMPLE_FACTORS           	-	qc2	group:qc | Sample source:pooled_qc	RAW_FILE_NAME(filename)=060-0145-QC_2_004.mzML
SUBJECT_SAMPLE_FACTORS           	-	qc3	group:qc | Sample source:pooled_qc	RAW_FILE_NAME(filename)=060-0145-QC_3_021.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Plasma samples were collected upon completion of the time course after fasting
CO:COLLECTION_SUMMARY            	the mice for 4 h.
CO:SAMPLE_TYPE                   	Blood (plasma)
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Mice were weight-matched and divided into two groups fed either HFD-control (60%
TR:TREATMENT_SUMMARY             	fat/0.86% methionine; Research Diets A14032002) or HFD-MetR (60% fat/0.12%
TR:TREATMENT_SUMMARY             	methionine; Research Diets A14032001). The HFD-MetR group was split into 3
TR:TREATMENT_SUMMARY             	groups, where the HFD-MetR was administered for 3 days (MetR3, n=8), 5 days
TR:TREATMENT_SUMMARY             	(MetR5, n=8), or 10 days (MetR10, n=7). HFD-MetR groups were compared to each
TR:TREATMENT_SUMMARY             	other and the control group (HFD, n=8).
TR:TREATMENT                     	Dietary intervention and duration of altered diet
TR:TREATMENT_ROUTE               	Diet
TR:TREATMENT_DOSE                	Research Diets A14032002; Research Diets A14032001
TR:TREATMENT_DOSEVOLUME          	HFD-control (60% fat/0.86% methionine; Research Diets A14032002) or HFD-MetR
TR:TREATMENT_DOSEVOLUME          	(60% fat/0.12% methionine; Research Diets A14032001)
TR:TREATMENT_DOSEDURATION        	3-10 days
TR:TREATMENT_VEHICLE             	Food
TR:ANIMAL_FASTING                	4h prior to sample collection
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	A 50 µL aliquot was transferred onto a solid phase extraction (SPE) system and
SP:SAMPLEPREP_SUMMARY            	10 µL was taken from each sample to form the pooled quality control sample. 200
SP:SAMPLEPREP_SUMMARY            	µL of 1:1 acetonitrile:methanol was added to each well and shaken for 1 min at
SP:SAMPLEPREP_SUMMARY            	room temperature at 360 rpm, followed by a 10 min incubation. Next, 150 µL of
SP:SAMPLEPREP_SUMMARY            	2:2:1 methanol:acetonitrile:water was added to each well and taken again for 10
SP:SAMPLEPREP_SUMMARY            	min. Polar metabolites were then eluted into the 96-well collection plate by
SP:SAMPLEPREP_SUMMARY            	using the positive pressure manifold. This is repeated with 100 µL of 2:2:1
SP:SAMPLEPREP_SUMMARY            	methanol:acetonitrile:water into the same collection plate. Polar eluates were
SP:SAMPLEPREP_SUMMARY            	then covered and stored at -80°C until LC/MS analysis. The SPE plates from the
SP:SAMPLEPREP_SUMMARY            	polar metabolite extraction were then washed twice with 500 µL 1:1 methyl
SP:SAMPLEPREP_SUMMARY            	tert-butyl ether:methanol to elute non-polar metabolites into a new collection
SP:SAMPLEPREP_SUMMARY            	plate using a positive pressure manifold. The combined eluates were dried under
SP:SAMPLEPREP_SUMMARY            	a stream of nitrogen at room temperature and reconstituted with 200 µL 1:1
SP:SAMPLEPREP_SUMMARY            	isopropanol:methanol and stored at -80°C prior to LC/MS analysis.
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACT_STORAGE               	-80℃
SP:SAMPLE_RESUSPENSION           	2:2:1 methanol:acetonitrile:water
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity II
CH:COLUMN_NAME                   	HILICON iHILIC (P)-Classic (100 x 2.1mm, 5um)
CH:SOLVENT_A                     	95% water/5% acetonitrile; 20mM ammonium bicarbonate; 0.1% ammonium hydroxide;
CH:SOLVENT_A                     	2.5 µM medronic acid
CH:SOLVENT_B                     	95% water/5% acetonitrile
CH:FLOW_GRADIENT                 	0-1 min: 90% B, 1-12 min: 90-35% B, 12-12.5 min: 35-20% B, 12.5 min-14.5 min:
CH:FLOW_GRADIENT                 	20% B
CH:FLOW_RATE                     	250 µL/min
CH:COLUMN_TEMPERATURE            	45
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6546 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	MS/MS data were acquired in a data-dependent iterative fashion with a 1.3 m/z
MS:MS_COMMENTS                   	isolation window. Features were detected in LC/MS data through in-house peak
MS:MS_COMMENTS                   	detection and curation software.
MS:MS_RESULTS_FILE               	ST004085_AN006769_Results.txt	UNITS:log2-transformed normalized relative abundance	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END