#METABOLOMICS WORKBENCH wpathmasiri_20210908_100810 DATATRACK_ID:2829 STUDY_ID:ST001914 ANALYSIS_ID:AN003112
VERSION                          	1
CREATED_ON                       	09-06-2023
#PROJECT
PR:PROJECT_TITLE                 	Fecal Metabolomics Reveals Products of Dysregulated Proteolysis and Altered
PR:PROJECT_TITLE                 	Microbial Metabolism in Obesity-Related Osteoarthritis
PR:PROJECT_TYPE                  	C18 Reversed-Phase Broad Spectrum Metabolomics
PR:PROJECT_SUMMARY               	Objective. The objective of this study was to determine if perturbations in gut
PR:PROJECT_SUMMARY               	microbial composition and the gut metabolome could be linked to individuals with
PR:PROJECT_SUMMARY               	obesity and osteoarthritis (OA). Methods. Fecal samples were collected from
PR:PROJECT_SUMMARY               	obese individuals diagnosed with radiographic hand plus knee OA (n=59), defined
PR:PROJECT_SUMMARY               	as involvement of at least 3 joints across both hands, and a Kellgren-Lawrence
PR:PROJECT_SUMMARY               	(KL) grade 2-4 (or total knee replacement) in at least one knee. Controls (n=33)
PR:PROJECT_SUMMARY               	were without hand OA and with KL grade 0-1 knees. Fecal metabolomes were
PR:PROJECT_SUMMARY               	analyzed by a UHPLC/Q Exactive HFx mass spectrometer. Microbiome composition was
PR:PROJECT_SUMMARY               	determined in fecal samples by 16S ribosomal RNA amplicon sequencing (rRNA-seq).
PR:PROJECT_SUMMARY               	Stepwise logistic regression models were built to determine microbiome and/or
PR:PROJECT_SUMMARY               	metabolic characteristics of OA. Results. Untargeted metabolomics analysis
PR:PROJECT_SUMMARY               	indicated that OA cases had significantly higher levels of di- and tri-peptides
PR:PROJECT_SUMMARY               	and significant perturbations in microbial metabolites including propionic acid,
PR:PROJECT_SUMMARY               	indoles and other tryptophan metabolites. Pathway analysis revealed several
PR:PROJECT_SUMMARY               	significantly perturbed pathways associated with OA including leukotriene
PR:PROJECT_SUMMARY               	metabolism, amino acid metabolism and fatty acid utilization. Logistic
PR:PROJECT_SUMMARY               	regression models selected metabolites associated with the gut microbiota and
PR:PROJECT_SUMMARY               	leaky gut syndrome as significant predictors of OA status, particularly when
PR:PROJECT_SUMMARY               	combined with the rRNA-seq data. Conclusions. Adults with obesity and OA have
PR:PROJECT_SUMMARY               	distinct fecal metabolomes characterized by increased products of proteolysis,
PR:PROJECT_SUMMARY               	perturbations in leukotriene metabolism, and changes in microbial metabolites
PR:PROJECT_SUMMARY               	compared with controls. These metabolic perturbations indicate a possible role
PR:PROJECT_SUMMARY               	of dysregulated proteolysis in OA.
PR:INSTITUTE                     	University of North Carolina at Chapel Hill
PR:DEPARTMENT                    	Medicine
PR:LABORATORY                    	UNC Thurston Arthritis Research Center, Division of Rheumatology, Allergy &
PR:LABORATORY                    	Immunology
PR:LAST_NAME                     	Loeser
PR:FIRST_NAME                    	Richard
PR:ADDRESS                       	3300 Thurston Building Campus Box 7280 Chapel Hill, NC 27599-7280
PR:EMAIL                         	richard_loeser@med.unc.edu
PR:PHONE                         	866-827-2862
PR:DOI                           	http://dx.doi.org/10.21228/M8PQ6M
#STUDY
ST:STUDY_TITLE                   	Fecal Metabolomics Reveals Products of Dysregulated Proteolysis and Altered
ST:STUDY_TITLE                   	Microbial Metabolism in Obesity-Related Osteoarthritis
ST:STUDY_TYPE                    	C18 Untargeted UPLC-MS Metabolomics Analysis
ST:STUDY_SUMMARY                 	Objective. The objective of this study was to determine if perturbations in gut
ST:STUDY_SUMMARY                 	microbial composition and the gut metabolome could be linked to individuals with
ST:STUDY_SUMMARY                 	obesity and osteoarthritis (OA). Methods. Fecal samples were collected from
ST:STUDY_SUMMARY                 	obese individuals diagnosed with radiographic hand plus knee OA (n=59), defined
ST:STUDY_SUMMARY                 	as involvement of at least 3 joints across both hands, and a Kellgren-Lawrence
ST:STUDY_SUMMARY                 	(KL) grade 2-4 (or total knee replacement) in at least one knee. Controls (n=33)
ST:STUDY_SUMMARY                 	were without hand OA and with KL grade 0-1 knees. Fecal metabolomes were
ST:STUDY_SUMMARY                 	analyzed by a UHPLC/Q Exactive HFx mass spectrometer. Microbiome composition was
ST:STUDY_SUMMARY                 	determined in fecal samples by 16S ribosomal RNA amplicon sequencing (rRNA-seq).
ST:STUDY_SUMMARY                 	Stepwise logistic regression models were built to determine microbiome and/or
ST:STUDY_SUMMARY                 	metabolic characteristics of OA. Results. Untargeted metabolomics analysis
ST:STUDY_SUMMARY                 	indicated that OA cases had significantly higher levels of di- and tri-peptides
ST:STUDY_SUMMARY                 	and significant perturbations in microbial metabolites including propionic acid,
ST:STUDY_SUMMARY                 	indoles and other tryptophan metabolites. Pathway analysis revealed several
ST:STUDY_SUMMARY                 	significantly perturbed pathways associated with OA including leukotriene
ST:STUDY_SUMMARY                 	metabolism, amino acid metabolism and fatty acid utilization. Logistic
ST:STUDY_SUMMARY                 	regression models selected metabolites associated with the gut microbiota and
ST:STUDY_SUMMARY                 	leaky gut syndrome as significant predictors of OA status, particularly when
ST:STUDY_SUMMARY                 	combined with the rRNA-seq data. Conclusions. Adults with obesity and OA have
ST:STUDY_SUMMARY                 	distinct fecal metabolomes characterized by increased products of proteolysis,
ST:STUDY_SUMMARY                 	perturbations in leukotriene metabolism, and changes in microbial metabolites
ST:STUDY_SUMMARY                 	compared with controls. These metabolic perturbations indicate a possible role
ST:STUDY_SUMMARY                 	of dysregulated proteolysis in OA.
ST:INSTITUTE                     	University of North Carolina at Chapel Hill
ST:DEPARTMENT                    	Nutrition
ST:LABORATORY                    	Metabolomics and Exposome Laboratory, Nutrition Research Institute, UNC Chapel
ST:LABORATORY                    	Hill
ST:LAST_NAME                     	Susan
ST:FIRST_NAME                    	Sumner
ST:ADDRESS                       	500 Laureate Way, Kannapolis, NC 28081
ST:EMAIL                         	susan_sumner@unc.edu
ST:PHONE                         	9196224456
ST:SUBMIT_DATE                   	2021-09-08
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:GENDER                        	Male and female
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	F_12	group:case | gender:F	RAW_FILE_NAME=F_12.raw
SUBJECT_SAMPLE_FACTORS           	-	F_13	group:case | gender:F	RAW_FILE_NAME=F_13.raw
SUBJECT_SAMPLE_FACTORS           	-	F_16	group:case | gender:F	RAW_FILE_NAME=F_16.raw
SUBJECT_SAMPLE_FACTORS           	-	F_18	group:case | gender:F	RAW_FILE_NAME=F_18.raw
SUBJECT_SAMPLE_FACTORS           	-	F_19	group:case | gender:F	RAW_FILE_NAME=F_19.raw
SUBJECT_SAMPLE_FACTORS           	-	F_20	group:case | gender:F	RAW_FILE_NAME=F_20.raw
SUBJECT_SAMPLE_FACTORS           	-	F_21	group:case | gender:F	RAW_FILE_NAME=F_21.raw
SUBJECT_SAMPLE_FACTORS           	-	F_22	group:case | gender:F	RAW_FILE_NAME=F_22.raw
SUBJECT_SAMPLE_FACTORS           	-	F_23	group:case | gender:F	RAW_FILE_NAME=F_23.raw
SUBJECT_SAMPLE_FACTORS           	-	F_26	group:case | gender:F	RAW_FILE_NAME=F_26.raw
SUBJECT_SAMPLE_FACTORS           	-	F_28	group:case | gender:F	RAW_FILE_NAME=F_28.raw
SUBJECT_SAMPLE_FACTORS           	-	F_29	group:case | gender:F	RAW_FILE_NAME=F_29.raw
SUBJECT_SAMPLE_FACTORS           	-	F_30	group:case | gender:F	RAW_FILE_NAME=F_30.raw
SUBJECT_SAMPLE_FACTORS           	-	F_31	group:case | gender:F	RAW_FILE_NAME=F_31.raw
SUBJECT_SAMPLE_FACTORS           	-	F_32	group:case | gender:F	RAW_FILE_NAME=F_32.raw
SUBJECT_SAMPLE_FACTORS           	-	F_33	group:case | gender:F	RAW_FILE_NAME=F_33.raw
SUBJECT_SAMPLE_FACTORS           	-	F_34	group:case | gender:F	RAW_FILE_NAME=F_34.raw
SUBJECT_SAMPLE_FACTORS           	-	F_35	group:case | gender:F	RAW_FILE_NAME=F_35.raw
SUBJECT_SAMPLE_FACTORS           	-	F_36	group:case | gender:F	RAW_FILE_NAME=F_36.raw
SUBJECT_SAMPLE_FACTORS           	-	F_37	group:case | gender:F	RAW_FILE_NAME=F_37.raw
SUBJECT_SAMPLE_FACTORS           	-	F_38	group:case | gender:F	RAW_FILE_NAME=F_38.raw
SUBJECT_SAMPLE_FACTORS           	-	F_40	group:case | gender:F	RAW_FILE_NAME=F_40.raw
SUBJECT_SAMPLE_FACTORS           	-	F_43	group:case | gender:F	RAW_FILE_NAME=F_43.raw
SUBJECT_SAMPLE_FACTORS           	-	F_44	group:case | gender:F	RAW_FILE_NAME=F_44.raw
SUBJECT_SAMPLE_FACTORS           	-	F_45	group:case | gender:F	RAW_FILE_NAME=F_45.raw
SUBJECT_SAMPLE_FACTORS           	-	F_47	group:case | gender:F	RAW_FILE_NAME=F_47.raw
SUBJECT_SAMPLE_FACTORS           	-	F_48	group:case | gender:F	RAW_FILE_NAME=F_48.raw
SUBJECT_SAMPLE_FACTORS           	-	F_49	group:case | gender:F	RAW_FILE_NAME=F_49.raw
SUBJECT_SAMPLE_FACTORS           	-	F_51	group:case | gender:F	RAW_FILE_NAME=F_51.raw
SUBJECT_SAMPLE_FACTORS           	-	F_53	group:case | gender:F	RAW_FILE_NAME=F_53.raw
SUBJECT_SAMPLE_FACTORS           	-	F_54	group:case | gender:F	RAW_FILE_NAME=F_54.raw
SUBJECT_SAMPLE_FACTORS           	-	F_55	group:case | gender:F	RAW_FILE_NAME=F_55.raw
SUBJECT_SAMPLE_FACTORS           	-	F_56	group:case | gender:F	RAW_FILE_NAME=F_56.raw
SUBJECT_SAMPLE_FACTORS           	-	F_58	group:case | gender:F	RAW_FILE_NAME=F_58.raw
SUBJECT_SAMPLE_FACTORS           	-	F_61	group:case | gender:F	RAW_FILE_NAME=F_61.raw
SUBJECT_SAMPLE_FACTORS           	-	F_62	group:case | gender:F	RAW_FILE_NAME=F_62.raw
SUBJECT_SAMPLE_FACTORS           	-	F_64	group:case | gender:F	RAW_FILE_NAME=F_64.raw
SUBJECT_SAMPLE_FACTORS           	-	F_65	group:case | gender:F	RAW_FILE_NAME=F_65.raw
SUBJECT_SAMPLE_FACTORS           	-	F_72	group:case | gender:F	RAW_FILE_NAME=F_72.raw
SUBJECT_SAMPLE_FACTORS           	-	F_74	group:case | gender:F	RAW_FILE_NAME=F_74.raw
SUBJECT_SAMPLE_FACTORS           	-	F_8	group:case | gender:F	RAW_FILE_NAME=F_8.raw
SUBJECT_SAMPLE_FACTORS           	-	F_84	group:case | gender:F	RAW_FILE_NAME=F_84.raw
SUBJECT_SAMPLE_FACTORS           	-	F_90	group:case | gender:F	RAW_FILE_NAME=F_90.raw
SUBJECT_SAMPLE_FACTORS           	-	F_24	group:case | gender:M	RAW_FILE_NAME=F_24.raw
SUBJECT_SAMPLE_FACTORS           	-	F_39	group:case | gender:M	RAW_FILE_NAME=F_39.raw
SUBJECT_SAMPLE_FACTORS           	-	F_41	group:case | gender:M	RAW_FILE_NAME=F_41.raw
SUBJECT_SAMPLE_FACTORS           	-	F_46	group:case | gender:M	RAW_FILE_NAME=F_46.raw
SUBJECT_SAMPLE_FACTORS           	-	F_57	group:case | gender:M	RAW_FILE_NAME=F_57.raw
SUBJECT_SAMPLE_FACTORS           	-	F_59	group:case | gender:M	RAW_FILE_NAME=F_59.raw
SUBJECT_SAMPLE_FACTORS           	-	F_63	group:case | gender:M	RAW_FILE_NAME=F_63.raw
SUBJECT_SAMPLE_FACTORS           	-	F_1	group:control | gender:F	RAW_FILE_NAME=F_1.raw
SUBJECT_SAMPLE_FACTORS           	-	F_11	group:control | gender:F	RAW_FILE_NAME=F_11.raw
SUBJECT_SAMPLE_FACTORS           	-	F_14	group:control | gender:F	RAW_FILE_NAME=F_14.raw
SUBJECT_SAMPLE_FACTORS           	-	F_15	group:control | gender:F	RAW_FILE_NAME=F_15.raw
SUBJECT_SAMPLE_FACTORS           	-	F_17	group:control | gender:F	RAW_FILE_NAME=F_17.raw
SUBJECT_SAMPLE_FACTORS           	-	F_25	group:control | gender:F	RAW_FILE_NAME=F_25.raw
SUBJECT_SAMPLE_FACTORS           	-	F_3	group:control | gender:F	RAW_FILE_NAME=F_3.raw
SUBJECT_SAMPLE_FACTORS           	-	F_4	group:control | gender:F	RAW_FILE_NAME=F_4.raw
SUBJECT_SAMPLE_FACTORS           	-	F_52	group:control | gender:F	RAW_FILE_NAME=F_52.raw
SUBJECT_SAMPLE_FACTORS           	-	F_6	group:control | gender:F	RAW_FILE_NAME=F_6.raw
SUBJECT_SAMPLE_FACTORS           	-	F_60	group:control | gender:F	RAW_FILE_NAME=F_60.raw
SUBJECT_SAMPLE_FACTORS           	-	F_67	group:control | gender:F	RAW_FILE_NAME=F_67.raw
SUBJECT_SAMPLE_FACTORS           	-	F_68	group:control | gender:F	RAW_FILE_NAME=F_68.raw
SUBJECT_SAMPLE_FACTORS           	-	F_69	group:control | gender:F	RAW_FILE_NAME=F_69.raw
SUBJECT_SAMPLE_FACTORS           	-	F_71	group:control | gender:F	RAW_FILE_NAME=F_71.raw
SUBJECT_SAMPLE_FACTORS           	-	F_73	group:control | gender:F	RAW_FILE_NAME=F_73.raw
SUBJECT_SAMPLE_FACTORS           	-	F_76	group:control | gender:F	RAW_FILE_NAME=F_76.raw
SUBJECT_SAMPLE_FACTORS           	-	F_78	group:control | gender:F	RAW_FILE_NAME=F_78.raw
SUBJECT_SAMPLE_FACTORS           	-	F_79	group:control | gender:F	RAW_FILE_NAME=F_79.raw
SUBJECT_SAMPLE_FACTORS           	-	F_80	group:control | gender:F	RAW_FILE_NAME=F_80.raw
SUBJECT_SAMPLE_FACTORS           	-	F_82	group:control | gender:F	RAW_FILE_NAME=F_82.raw
SUBJECT_SAMPLE_FACTORS           	-	F_85	group:control | gender:F	RAW_FILE_NAME=F_85.raw
SUBJECT_SAMPLE_FACTORS           	-	F_86	group:control | gender:F	RAW_FILE_NAME=F_86.raw
SUBJECT_SAMPLE_FACTORS           	-	F_89	group:control | gender:F	RAW_FILE_NAME=F_89.raw
SUBJECT_SAMPLE_FACTORS           	-	F_91	group:control | gender:F	RAW_FILE_NAME=F_91.raw
SUBJECT_SAMPLE_FACTORS           	-	F_92	group:control | gender:F	RAW_FILE_NAME=F_92.raw
SUBJECT_SAMPLE_FACTORS           	-	F_10	group:control | gender:M	RAW_FILE_NAME=F_10.raw
SUBJECT_SAMPLE_FACTORS           	-	F_2	group:control | gender:M	RAW_FILE_NAME=F_2.raw
SUBJECT_SAMPLE_FACTORS           	-	F_27	group:control | gender:M	RAW_FILE_NAME=F_27.raw
SUBJECT_SAMPLE_FACTORS           	-	F_42	group:control | gender:M	RAW_FILE_NAME=F_42.raw
SUBJECT_SAMPLE_FACTORS           	-	F_5	group:control | gender:M	RAW_FILE_NAME=F_5.raw
SUBJECT_SAMPLE_FACTORS           	-	F_50	group:control | gender:M	RAW_FILE_NAME=F_50.raw
SUBJECT_SAMPLE_FACTORS           	-	F_66	group:control | gender:M	RAW_FILE_NAME=F_66.raw
SUBJECT_SAMPLE_FACTORS           	-	F_7	group:control | gender:M	RAW_FILE_NAME=F_7.raw
SUBJECT_SAMPLE_FACTORS           	-	F_70	group:control | gender:M	RAW_FILE_NAME=F_70.raw
SUBJECT_SAMPLE_FACTORS           	-	F_75	group:control | gender:M	RAW_FILE_NAME=F_75.raw
SUBJECT_SAMPLE_FACTORS           	-	F_77	group:control | gender:M	RAW_FILE_NAME=F_77.raw
SUBJECT_SAMPLE_FACTORS           	-	F_81	group:control | gender:M	RAW_FILE_NAME=F_81.raw
SUBJECT_SAMPLE_FACTORS           	-	F_83	group:control | gender:M	RAW_FILE_NAME=F_83.raw
SUBJECT_SAMPLE_FACTORS           	-	F_87	group:control | gender:M	RAW_FILE_NAME=F_87.raw
SUBJECT_SAMPLE_FACTORS           	-	F_88	group:control | gender:M	RAW_FILE_NAME=F_88.raw
SUBJECT_SAMPLE_FACTORS           	-	F_9	group:control | gender:M	RAW_FILE_NAME=F_9.raw
SUBJECT_SAMPLE_FACTORS           	-	SP_1	group:quality control pool | gender:-	RAW_FILE_NAME=SP_1.raw
SUBJECT_SAMPLE_FACTORS           	-	SP_2	group:quality control pool | gender:-	RAW_FILE_NAME=SP_2.raw
SUBJECT_SAMPLE_FACTORS           	-	SP_3	group:quality control pool | gender:-	RAW_FILE_NAME=SP_3.raw
SUBJECT_SAMPLE_FACTORS           	-	SP_4	group:quality control pool | gender:-	RAW_FILE_NAME=SP_4.raw
SUBJECT_SAMPLE_FACTORS           	-	SP_5	group:quality control pool | gender:-	RAW_FILE_NAME=SP_5.raw
SUBJECT_SAMPLE_FACTORS           	-	SP_6	group:quality control pool | gender:-	RAW_FILE_NAME=SP_6.raw
SUBJECT_SAMPLE_FACTORS           	-	SP_7	group:quality control pool | gender:-	RAW_FILE_NAME=SP_7.raw
SUBJECT_SAMPLE_FACTORS           	-	SP_8	group:quality control pool | gender:-	RAW_FILE_NAME=SP_8.raw
SUBJECT_SAMPLE_FACTORS           	-	SP_9	group:quality control pool | gender:-	RAW_FILE_NAME=SP_9.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Fecal samples were collected at each participant's home in and placed in a
CO:COLLECTION_SUMMARY            	plastic biohazard bag with a frozen icepack to keep the samples cold. The
CO:COLLECTION_SUMMARY            	samples were picked up by study personnel or returned to the study clinic by the
CO:COLLECTION_SUMMARY            	participant within 24 hours of the time the stool was collected. Stool samples
CO:COLLECTION_SUMMARY            	were stored at the clinic site in a -200C freezer for up to one week until
CO:COLLECTION_SUMMARY            	transfer to a -800C freezer where samples were kept until processing.
CO:SAMPLE_TYPE                   	Feces
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	NA
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Ninety-two fecal samples (250-300 mg) were randomized and homogenized in 50:50
SP:SAMPLEPREP_SUMMARY            	acetonitrile:water (5 mL/ mg fecal mass) in MagNALyser tubes with ceramic beads,
SP:SAMPLEPREP_SUMMARY            	using an Omni Bead Ruptor (5 meters per second, two 30-sec cycles, 15 sec dwell
SP:SAMPLEPREP_SUMMARY            	time in between cycles). Samples were centrifuged at 16,000 relative centrifugal
SP:SAMPLEPREP_SUMMARY            	force (rcf) for 15 min, the supernatant was transferred, and centrifuged again
SP:SAMPLEPREP_SUMMARY            	at 16,000 rcf for 5 min. Quality control (QC) samples were prepared by pooling
SP:SAMPLEPREP_SUMMARY            	70 µL supernatant from each of the study samples and processed identically to
SP:SAMPLEPREP_SUMMARY            	the study samples. An aliquot of the supernatant (100 mL) from each sample was
SP:SAMPLEPREP_SUMMARY            	transferred to 2.0 mL tubes and dried overnight by SpeedVac and then
SP:SAMPLEPREP_SUMMARY            	reconstituted in 200 mL of reconstitution solution (95:5 water-methanol solvent
SP:SAMPLEPREP_SUMMARY            	containing 500 ng/ml tryptophan d-5). Samples were centrifuged at 16,000 rcf at
SP:SAMPLEPREP_SUMMARY            	4°C for 4 minutes and the supernatants were transferred to autosampler vials.
SP:SAMPLEPREP_SUMMARY            	The study samples were randomized with interspersed QC pools before data
SP:SAMPLEPREP_SUMMARY            	acquisition. An injection volume of 5 mL was used for the UPLC-MS analysis.
SP:SAMPLEPREP_PROTOCOL_FILENAME  	OA-Fecal LCMS procedures
SP:PROCESSING_METHOD             	Extraction
SP:PROCESSING_STORAGE_CONDITIONS 	On ice
SP:EXTRACT_STORAGE               	-80℃
SP:SAMPLE_RESUSPENSION           	95:5 water-methanol solvent containing 500 ng/ml tryptophan d-5
#CHROMATOGRAPHY
CH:INSTRUMENT_NAME               	none
CH:COLUMN_NAME                   	none
CH:COLUMN_PRESSURE               	6000-10000
CH:COLUMN_TEMPERATURE            	50
CH:FLOW_GRADIENT                 	Time(min) Flow Rate %A %B Curve 1. 0 0.4 99.0 1.0 5 2. 1.00 0.4 99.0 1.0 5 3.
CH:FLOW_GRADIENT                 	16.00 0.4 1.0 99.0 5 4. 19.00 0.4 1.0 99.0 5 5. 19.50 0.4 99.0 1.0 5
CH:FLOW_RATE                     	0.4 mL/min
CH:INJECTION_TEMPERATURE         	8
CH:INTERNAL_STANDARD             	Tryptophan-d5
CH:SOLVENT_A                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% methanol; 0.1% formic acid
CH:ANALYTICAL_TIME               	22 min
CH:WEAK_WASH_SOLVENT_NAME        	10:90 Methanol:Water with 0.1% FA solution
CH:STRONG_WASH_SOLVENT_NAME      	75:25 2-Propanol: Water with 0.1% FA solution
CH:RANDOMIZATION_ORDER           	Yes
CH:CHROMATOGRAPHY_TYPE           	Unspecified
#ANALYSIS
AN:LABORATORY_NAME               	UNC-NRI Metabolomics and Exposome Laboratory
AN:ANALYSIS_TYPE                 	MS
AN:ACQUISITION_DATE              	12/7/2018 through 12/9/2018
AN:SOFTWARE_VERSION              	Xcalibur 4.1
AN:OPERATOR_NAME                 	Yuanyuan Li
AN:DETECTOR_TYPE                 	Orbitrap
AN:DATA_FORMAT                   	profile
#MS
MS:INSTRUMENT_NAME               	Orbitrap Q-Exactive HF-X
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	We used DDA mode to acquire the MS and MS/MS data. Progenesis QI was used for
MS:MS_COMMENTS                   	peak picking, alignment, and normalization.
MS:ION_MODE                      	POSITIVE
MS:CAPILLARY_TEMPERATURE         	275 °C
MS:CAPILLARY_VOLTAGE             	3.5 KV
MS:COLLISION_ENERGY              	10-35, ramp
MS:COLLISION_GAS                 	N2
MS:DRY_GAS_FLOW                  	45
MS:DRY_GAS_TEMP                  	325°C
MS:FRAGMENTATION_METHOD          	CID
MS:MASS_ACCURACY                 	5 ppm
MS:DESOLVATION_GAS_FLOW          	45
MS:DESOLVATION_TEMPERATURE       	325°C
MS:MS_RESULTS_FILE               	ST001914_AN003112_Results.txt	UNITS:Normalized intensity	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END