VERSION             	1
CREATED_ON             	July 31, 2023, 7:52 am
PR:PROJECT_TITLE                 	Nontargeted Serum Metabolomic Profiling of FXR-null and Wild-type Mice
PR:PROJECT_SUMMARY               	This study analyzed the serum metabolome of 10 FXR-knockout and 10 wild-type
PR:PROJECT_SUMMARY               	mice by RPLC-HRMS.
PR:INSTITUTE                     	University of North Carolina at Chapel Hill
PR:DEPARTMENT                    	Department of Environmental Sciences and Engineering
PR:LABORATORY                    	Kun Lu
PR:LAST_NAME                     	Hsiao
PR:FIRST_NAME                    	Yun-Chung
PR:ADDRESS                       	135 Dauer Dr. MHRC1104
PR:EMAIL                         	ychsiao@live.unc.edu
PR:PHONE                         	9842159592
PR:FUNDING_SOURCE                	The research was supported by the UNC Superfund Research program (P42ES031007),
PR:FUNDING_SOURCE                	University of North Carolina Center for Environmental Health and Susceptibility
PR:FUNDING_SOURCE                	grant (P30ES010126).
ST:STUDY_TITLE                   	Nontargeted Serum Metabolomic Profiling of FXR-null and Wild-type Mice
ST:STUDY_SUMMARY                 	This study analyzed the serum metabolome of 10 FXR-knockout and 10 wild-type
ST:STUDY_SUMMARY                 	mice by RPLC-HRMS.
ST:INSTITUTE                     	University of North Carolina at Chapel Hill
ST:DEPARTMENT                    	Department of Environmental Sciences and Engineering
ST:LABORATORY                    	Kun Lu
ST:LAST_NAME                     	Hsiao
ST:FIRST_NAME                    	Yun-Chung
ST:ADDRESS                       	135 Dauer Dr. MHRC1104
ST:EMAIL                         	ychsiao@live.unc.edu
ST:PHONE                         	9842159592
ST:NUM_GROUPS                    	2
ST:TOTAL_SUBJECTS                	20
ST:NUM_MALES                     	20
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:AGE_OR_AGE_RANGE              	6 months
SU:GENDER                        	Male
SU:ANIMAL_ANIMAL_SUPPLIER        	The Jackson Laboratory
#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           	-	G31-Con-31A1A	Genotype:Wild-type	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO4_Sample_G31-Con-31A1A_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G31-Con-31A1B	Genotype:Wild-type	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO8_Sample_G31-Con-31A1B_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G31-Con-31A2A	Genotype:Wild-type	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO15_Sample_G31-Con-31A2A_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G31-Con-31A2B	Genotype:Wild-type	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO19_Sample_G31-Con-31A2B_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G31-Con-31A3A	Genotype:Wild-type	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO26_Sample_G31-Con-31A3A_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G31-Con-31A3B	Genotype:Wild-type	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO30_Sample_G31-Con-31A3B_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G31-Con-31A4A	Genotype:Wild-type	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO37_Sample_G31-Con-31A4A_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G31-Con-31A4B	Genotype:Wild-type	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO41_Sample_G31-Con-31A4B_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G31-Con-31A5A	Genotype:Wild-type	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO48_Sample_G31-Con-31A5A_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G31-Con-31A5B	Genotype:Wild-type	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO52_Sample_G31-Con-31A5B_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G32-Con-32A1A	Genotype:FXR-knockout	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO6_Sample_G32-Con-32A1A_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G32-Con-32A1B	Genotype:FXR-knockout	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO10_Sample_G32-Con-32A1B_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G32-Con-32A2A	Genotype:FXR-knockout	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO17_Sample_G32-Con-32A2A_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G32-Con-32A2B	Genotype:FXR-knockout	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO21_Sample_G32-Con-32A2B_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G32-Con-32A5A	Genotype:FXR-knockout	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO28_Sample_G32-Con-32A5A_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G32-Con-32A5B	Genotype:FXR-knockout	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO32_Sample_G32-Con-32A5B_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G32-Con-32A6A	Genotype:FXR-knockout	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO39_Sample_G32-Con-32A6A_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G32-Con-32A6B	Genotype:FXR-knockout	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO43_Sample_G32-Con-32A6B_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G32-Con-32A8A	Genotype:FXR-knockout	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO50_Sample_G32-Con-32A8A_10uL.raw
SUBJECT_SAMPLE_FACTORS           	-	G32-Con-32A8B	Genotype:FXR-knockout	RAW_FILE_NAME=T3_400uL_pos_20230711_serum_IO54_Sample_G32-Con-32A8B_10uL.raw
CO:COLLECTION_SUMMARY            	Serum samples from each mouse were collected from heart blood immediately after
CO:COLLECTION_SUMMARY            	carbon dioxide euthanasia.
CO:SAMPLE_TYPE                   	Blood (serum)
TR:TREATMENT_SUMMARY             	No treatment.
SP:SAMPLEPREP_SUMMARY            	Serum samples from each mouse were thawed and 20 μL was consumed to extract
SP:SAMPLEPREP_SUMMARY            	metabolites for each mice. Metabolite extraction was facilitated by adding 180
SP:SAMPLEPREP_SUMMARY            	μL methanol containing stable isotope-labeled chemicals ([D5]-glutamine,
SP:SAMPLEPREP_SUMMARY            	[D2]-𝛾-aminobutyric acid, [D3]-tryptophan, [D2]-indole-3-propionic acid,
SP:SAMPLEPREP_SUMMARY            	[D2]-indole-3-acetic acid, [D13]-acetylcholine in 500 nM; [D4]-serotonin in 250
SP:SAMPLEPREP_SUMMARY            	nM; and [D4]-kynurenic acid in 50 nM), and incubated under -20°C for 1 hr,
SP:SAMPLEPREP_SUMMARY            	followed by centrifugation under 15,000 ×g, 4°C for 10 minutes to collect the
SP:SAMPLEPREP_SUMMARY            	supernatant (150 μL). The supernatant was dried by SpeedVac® and reconstituted
SP:SAMPLEPREP_SUMMARY            	with 100 μL 2% acetonitrile in water.
CH:CHROMATOGRAPHY_SUMMARY        	The serum analytes were injected (10 μL) into a Waters Acquity UPLC HSS T3
CH:CHROMATOGRAPHY_SUMMARY        	(reverse phase C18, 100 Å, 1.8 μm, 2.1 mm × 100 mm) analytical column
CH:CHROMATOGRAPHY_SUMMARY        	controlled at 40 °C, with the mobile phase composed of water (A) and
CH:CHROMATOGRAPHY_SUMMARY        	acetonitrile (B) both added with 0.1% formic acid at a flow rate of 0.4 mL/min.
CH:CHROMATOGRAPHY_SUMMARY        	The 15-min-gradient for chromatographic separation was set as the following: 2%
CH:CHROMATOGRAPHY_SUMMARY        	B from 0-1 min; 2%-15% B from 1-3 min; 15%-50% B from 3-6 min; 50%-98% B from
CH:CHROMATOGRAPHY_SUMMARY        	6-7.5 min; 98% B held from 7.5-11.5 min; 98%-2% B from 11.5-11.6 min; and 2% B
CH:CHROMATOGRAPHY_SUMMARY        	held from 11.6-15 min for a final re-equilibration.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um)
CH:SOLVENT_A                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% acetonitrile; 0.1% formic acid
CH:FLOW_GRADIENT                 	2% B from 0-1 min; 2%-15% B from 1-3 min; 15%-50% B from 3-6 min; 50%-98% B from
CH:FLOW_GRADIENT                 	6-7.5 min; 98% B held from 7.5-11.5 min; 98%-2% B from 11.5-11.6 min; and 2% B
CH:FLOW_GRADIENT                 	held from 11.6-15 min for a final re-equilibration.
CH:FLOW_RATE                     	0.4 mL/min
AN:ANALYSIS_TYPE                 	MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The mass spectrometry was set to scan under the positive mode with the sheath
MS:MS_COMMENTS                   	gas, auxiliary gas, and sweep gas set to flow rates of 50, 13, and 3 psi,
MS:MS_COMMENTS                   	respectively. With the spray voltage set to 3.5 kV, the capillary and auxiliary
MS:MS_COMMENTS                   	gas heating temperature were respectively controlled to 263°C and 425°C to
MS:MS_COMMENTS                   	fully scan across m/z 70 to 1,000. The resolution was set to 70,000 FWHM (m/z
MS:MS_COMMENTS                   	200). The automatic gain control (AGC) and the maximal injection time (MIT) was
MS:MS_COMMENTS                   	set to 2×105 and 50 msec, respectively. Routine mass calibrations were
MS:MS_COMMENTS                   	conducted before and after the sample analysis. The samples were
MS:MS_COMMENTS                   	blocked-randomized for the injection order. Quality control samples for the
MS:MS_COMMENTS                   	serum analytes were prepared by pooling the aliquots of each sample. Method
MS:MS_COMMENTS                   	blank samples were prepared for the serum samples by surrogating the biospecimen
MS:MS_COMMENTS                   	with water and following the same experimental procedures. If MS/MS spectrums
MS:MS_COMMENTS                   	were to be collected, the parallel reaction monitoring (PRM) mode was used, with
MS:MS_COMMENTS                   	the isolation width, AGC, and MIT set to 1.2, 3×105 and 100 msec, respectively,
MS:MS_COMMENTS                   	at the resolution of 17,500 FWHM (m/z 200).
MS:MS_RESULTS_FILE               	ST002802_AN004558_Results.txt	UNITS:m/z	Has m/z:Yes	Has RT:Yes	RT units:Minutes