#METABOLOMICS WORKBENCH Mahamogren_20230911_215002 DATATRACK_ID:4305 STUDY_ID:ST002934 ANALYSIS_ID:AN004813
VERSION                          	1
CREATED_ON                       	10-17-2023
#PROJECT
PR:PROJECT_TITLE                 	Metabolic profiling of newborn DBS samples associated with transit and false
PR:PROJECT_TITLE                 	elevation of glutarylcarnitine (C5DC)
PR:PROJECT_SUMMARY               	Background: Glutaric aciduria type-1 (GA-1) is a rare autosomal recessive
PR:PROJECT_SUMMARY               	metabolic disorder caused by a glutaryl coenzyme A dehydrogenase (GCDH)
PR:PROJECT_SUMMARY               	deficiency, affecting approximately 1 in 110,000 individuals globally. This
PR:PROJECT_SUMMARY               	enzymatic deficiency leads to abnormal elevations of glutaryl-CoA and its
PR:PROJECT_SUMMARY               	derivatives, specifically glutaric acid (GA), 3-hydroxyglutaric acid (3OHGA),
PR:PROJECT_SUMMARY               	and glutarylcarnitine (C5DC). Clinical manifestations encompass macrocephaly,
PR:PROJECT_SUMMARY               	developmental delays, and movement disorders. Early detection via genetic
PR:PROJECT_SUMMARY               	testing and newborn screening (NBS), utilizing GA-1 biomarkers in dried blood
PR:PROJECT_SUMMARY               	spot (DBS) samples, is vital for prompt intervention. Despite the NBS system,
PR:PROJECT_SUMMARY               	transit-elevated C5DC-containing DBS samples from falsely suspected GA-1
PR:PROJECT_SUMMARY               	newborns sometimes yield normal results, posing diagnostic sensitivity and
PR:PROJECT_SUMMARY               	specificity challenges. Consequently, there is a growing need for alternative
PR:PROJECT_SUMMARY               	diagnostic tools. Comprehensive mass spectrometry-based untargeted metabolomics
PR:PROJECT_SUMMARY               	offers promise in identifying additional informative biomarkers for
PR:PROJECT_SUMMARY               	distinguishing falsely suspected GA-1 newborns from healthy counterparts.
PR:PROJECT_SUMMARY               	Methodology: In this prospective study, we obtained DBS samples with
PR:PROJECT_SUMMARY               	transit-elevated C5DC levels from falsely suspected GA-1 newborns (n=47) and
PR:PROJECT_SUMMARY               	matched control DBS samples from healthy newborns (n=47) through the NBS
PR:PROJECT_SUMMARY               	program. Metabolites were extracted and analyzed via liquid
PR:PROJECT_SUMMARY               	chromatography-high-resolution mass spectrometry (LC-HRMS). Subsequent
PR:PROJECT_SUMMARY               	multivariate and univariate statistical analyses and feature annotation enabled
PR:PROJECT_SUMMARY               	biomarker and pathway investigations for significantly altered metabolites.
PR:PROJECT_SUMMARY               	Results: Untargeted metabolomics analysis revealed alterations in 582
PR:PROJECT_SUMMARY               	upregulated and 546 downregulated metabolites. The commonly used GA-1
PR:PROJECT_SUMMARY               	biomarkers, including C5DC, exhibited no significant changes in the falsely
PR:PROJECT_SUMMARY               	suspected GA-1 DBS samples. Conversely, 155 endogenous metabolites displayed
PR:PROJECT_SUMMARY               	significant variations compared to the control group. Furthermore, our data
PR:PROJECT_SUMMARY               	identified novel altered metabolic biomarkers, such as N-Palmitoylcysteine,
PR:PROJECT_SUMMARY               	3-hydroxylinoleoylcarnitine, Heptacarboxyporphyrin, and MG (0:0/20:1/0:0), along
PR:PROJECT_SUMMARY               	with perturbed metabolic pathways like sphingolipid and thiamine metabolism
PR:PROJECT_SUMMARY               	associated with the transient and falsely elevated C5DC levels in DBS samples.
PR:PROJECT_SUMMARY               	Conclusions: Our untargeted metabolomics investigation unveiled distinct
PR:PROJECT_SUMMARY               	metabolic pathways and biomarkers linked to the transient C5DC elevation in DBS
PR:PROJECT_SUMMARY               	samples from falsely suspected GA-1 newborns. These findings can enhance GA-1
PR:PROJECT_SUMMARY               	diagnosis by serving as predictive indicators during NBS analysis. Validation
PR:PROJECT_SUMMARY               	studies are warranted to confirm the presence of these newly identified
PR:PROJECT_SUMMARY               	metabolic pathways and biomarkers in confirmed GA-1 cases.
PR:INSTITUTE                     	King Faisal Specialist Hospital and Research Centre (KFSHRC)
PR:LAST_NAME                     	Al Mogren
PR:FIRST_NAME                    	Maha
PR:ADDRESS                       	Zahrawi Street, Al Maather, Riyadh 11211, Saudi Arabia
PR:EMAIL                         	mahamogren@gmail.com
PR:PHONE                         	966541205332
PR:DOI                           	http://dx.doi.org/10.21228/M8T714
#STUDY
ST:STUDY_TITLE                   	Metabolic profiling of newborn DBS samples associated with transit and false
ST:STUDY_TITLE                   	elevation of glutarylcarnitine (C5DC)
ST:STUDY_SUMMARY                 	Background: Glutaric aciduria type-1 (GA-1) is a rare autosomal recessive
ST:STUDY_SUMMARY                 	metabolic disorder caused by a glutaryl coenzyme A dehydrogenase (GCDH)
ST:STUDY_SUMMARY                 	deficiency, affecting approximately 1 in 110,000 individuals globally. This
ST:STUDY_SUMMARY                 	enzymatic deficiency leads to abnormal elevations of glutaryl-CoA and its
ST:STUDY_SUMMARY                 	derivatives, specifically glutaric acid (GA), 3-hydroxyglutaric acid (3OHGA),
ST:STUDY_SUMMARY                 	and glutarylcarnitine (C5DC). Clinical manifestations encompass macrocephaly,
ST:STUDY_SUMMARY                 	developmental delays, and movement disorders. Early detection via genetic
ST:STUDY_SUMMARY                 	testing and newborn screening (NBS), utilizing GA-1 biomarkers in dried blood
ST:STUDY_SUMMARY                 	spot (DBS) samples, is vital for prompt intervention. Despite the NBS system,
ST:STUDY_SUMMARY                 	transit-elevated C5DC-containing DBS samples from falsely suspected GA-1
ST:STUDY_SUMMARY                 	newborns sometimes yield normal results, posing diagnostic sensitivity and
ST:STUDY_SUMMARY                 	specificity challenges. Consequently, there is a growing need for alternative
ST:STUDY_SUMMARY                 	diagnostic tools. Comprehensive mass spectrometry-based untargeted metabolomics
ST:STUDY_SUMMARY                 	offers promise in identifying additional informative biomarkers for
ST:STUDY_SUMMARY                 	distinguishing falsely suspected GA-1 newborns from healthy counterparts.
ST:STUDY_SUMMARY                 	Methodology: In this prospective study, we obtained DBS samples with
ST:STUDY_SUMMARY                 	transit-elevated C5DC levels from falsely suspected GA-1 newborns (n=47) and
ST:STUDY_SUMMARY                 	matched control DBS samples from healthy newborns (n=47) through the NBS
ST:STUDY_SUMMARY                 	program. Metabolites were extracted and analyzed via liquid
ST:STUDY_SUMMARY                 	chromatography-high-resolution mass spectrometry (LC-HRMS). Subsequent
ST:STUDY_SUMMARY                 	multivariate and univariate statistical analyses and feature annotation enabled
ST:STUDY_SUMMARY                 	biomarker and pathway investigations for significantly altered metabolites.
ST:STUDY_SUMMARY                 	Results: Untargeted metabolomics analysis revealed alterations in 582
ST:STUDY_SUMMARY                 	upregulated and 546 downregulated metabolites. The commonly used GA-1
ST:STUDY_SUMMARY                 	biomarkers, including C5DC, exhibited no significant changes in the falsely
ST:STUDY_SUMMARY                 	suspected GA-1 DBS samples. Conversely, 155 endogenous metabolites displayed
ST:STUDY_SUMMARY                 	significant variations compared to the control group. Furthermore, our data
ST:STUDY_SUMMARY                 	identified novel altered metabolic biomarkers, such as N-Palmitoylcysteine,
ST:STUDY_SUMMARY                 	3-hydroxylinoleoylcarnitine, Heptacarboxyporphyrin, and MG (0:0/20:1/0:0), along
ST:STUDY_SUMMARY                 	with perturbed metabolic pathways like sphingolipid and thiamine metabolism
ST:STUDY_SUMMARY                 	associated with the transient and falsely elevated C5DC levels in DBS samples.
ST:STUDY_SUMMARY                 	Conclusions: Our untargeted metabolomics investigation unveiled distinct
ST:STUDY_SUMMARY                 	metabolic pathways and biomarkers linked to the transient C5DC elevation in DBS
ST:STUDY_SUMMARY                 	samples from falsely suspected GA-1 newborns. These findings can enhance GA-1
ST:STUDY_SUMMARY                 	diagnosis by serving as predictive indicators during NBS analysis. Validation
ST:STUDY_SUMMARY                 	studies are warranted to confirm the presence of these newly identified
ST:STUDY_SUMMARY                 	metabolic pathways and biomarkers in confirmed GA-1 cases.
ST:INSTITUTE                     	King Faisal Specialist Hospital and Research Centre (KFSHRC)
ST:LAST_NAME                     	Al Mogren
ST:FIRST_NAME                    	Maha
ST:ADDRESS                       	Zahrawi Street, Al Maather, Riyadh 11211, Saudi Arabia
ST:EMAIL                         	mahamogren@gmail.com
ST:PHONE                         	966541205332
ST:SUBMIT_DATE                   	2023-09-11
#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           	-	19281533_C	Factor:Control	RAW_FILE_NAME=19281533_C
SUBJECT_SAMPLE_FACTORS           	-	19534501_C	Factor:Control	RAW_FILE_NAME=19534501_C
SUBJECT_SAMPLE_FACTORS           	-	20419817_C	Factor:Control	RAW_FILE_NAME=20419817_C
SUBJECT_SAMPLE_FACTORS           	-	20649302_C	Factor:Control	RAW_FILE_NAME=20649302_C
SUBJECT_SAMPLE_FACTORS           	-	20688684_C	Factor:Control	RAW_FILE_NAME=20688684_C
SUBJECT_SAMPLE_FACTORS           	-	20688815_C	Factor:Control	RAW_FILE_NAME=20688815_C
SUBJECT_SAMPLE_FACTORS           	-	20771984_C	Factor:Control	RAW_FILE_NAME=20771984_C
SUBJECT_SAMPLE_FACTORS           	-	20799041_C	Factor:Control	RAW_FILE_NAME=20799041_C
SUBJECT_SAMPLE_FACTORS           	-	20799050_C	Factor:Control	RAW_FILE_NAME=20799050_C
SUBJECT_SAMPLE_FACTORS           	-	20799892_C	Factor:Control	RAW_FILE_NAME=20799892_C
SUBJECT_SAMPLE_FACTORS           	-	20799980_C	Factor:Control	RAW_FILE_NAME=20799980_C
SUBJECT_SAMPLE_FACTORS           	-	20808598_C	Factor:Control	RAW_FILE_NAME=20808598_C
SUBJECT_SAMPLE_FACTORS           	-	20808604_C	Factor:Control	RAW_FILE_NAME=20808604_C
SUBJECT_SAMPLE_FACTORS           	-	20808613_C	Factor:Control	RAW_FILE_NAME=20808613_C
SUBJECT_SAMPLE_FACTORS           	-	20809384_C	Factor:Control	RAW_FILE_NAME=20809384_C
SUBJECT_SAMPLE_FACTORS           	-	20818595_C	Factor:Control	RAW_FILE_NAME=20818595_C
SUBJECT_SAMPLE_FACTORS           	-	20820305_C	Factor:Control	RAW_FILE_NAME=20820305_C
SUBJECT_SAMPLE_FACTORS           	-	20830117_C	Factor:Control	RAW_FILE_NAME=20830117_C
SUBJECT_SAMPLE_FACTORS           	-	20830418_C	Factor:Control	RAW_FILE_NAME=20830418_C
SUBJECT_SAMPLE_FACTORS           	-	20839215_C	Factor:Control	RAW_FILE_NAME=20839215_C
SUBJECT_SAMPLE_FACTORS           	-	20851147_C	Factor:Control	RAW_FILE_NAME=20851147_C
SUBJECT_SAMPLE_FACTORS           	-	20851217_C	Factor:Control	RAW_FILE_NAME=20851217_C
SUBJECT_SAMPLE_FACTORS           	-	20859015_C	Factor:Control	RAW_FILE_NAME=20859015_C
SUBJECT_SAMPLE_FACTORS           	-	20859945_C	Factor:Control	RAW_FILE_NAME=20859945_C
SUBJECT_SAMPLE_FACTORS           	-	20859954_C	Factor:Control	RAW_FILE_NAME=20859954_C
SUBJECT_SAMPLE_FACTORS           	-	20864956_C	Factor:Control	RAW_FILE_NAME=20864956_C
SUBJECT_SAMPLE_FACTORS           	-	20882846_C	Factor:Control	RAW_FILE_NAME=20882846_C
SUBJECT_SAMPLE_FACTORS           	-	20884792_C	Factor:Control	RAW_FILE_NAME=20884792_C
SUBJECT_SAMPLE_FACTORS           	-	20890216_C	Factor:Control	RAW_FILE_NAME=20890216_C
SUBJECT_SAMPLE_FACTORS           	-	20902618_C	Factor:Control	RAW_FILE_NAME=20902618_C
SUBJECT_SAMPLE_FACTORS           	-	20940654_C	Factor:Control	RAW_FILE_NAME=20940654_C
SUBJECT_SAMPLE_FACTORS           	-	20975067_C	Factor:Control	RAW_FILE_NAME=20975067_C
SUBJECT_SAMPLE_FACTORS           	-	21027899_C	Factor:Control	RAW_FILE_NAME=21027899_C
SUBJECT_SAMPLE_FACTORS           	-	21073931_C	Factor:Control	RAW_FILE_NAME=21073931_C
SUBJECT_SAMPLE_FACTORS           	-	21077520_C	Factor:Control	RAW_FILE_NAME=21077520_C
SUBJECT_SAMPLE_FACTORS           	-	21462162_C	Factor:Control	RAW_FILE_NAME=21462162_C
SUBJECT_SAMPLE_FACTORS           	-	21484386_C	Factor:Control	RAW_FILE_NAME=21484386_C
SUBJECT_SAMPLE_FACTORS           	-	21492242_C	Factor:Control	RAW_FILE_NAME=21492242_C
SUBJECT_SAMPLE_FACTORS           	-	21501966_C	Factor:Control	RAW_FILE_NAME=21501966_C
SUBJECT_SAMPLE_FACTORS           	-	21554348_C	Factor:Control	RAW_FILE_NAME=21554348_C
SUBJECT_SAMPLE_FACTORS           	-	21554366_C	Factor:Control	RAW_FILE_NAME=21554366_C
SUBJECT_SAMPLE_FACTORS           	-	21558089_C	Factor:Control	RAW_FILE_NAME=21558089_C
SUBJECT_SAMPLE_FACTORS           	-	21610033_C	Factor:Control	RAW_FILE_NAME=21610033_C
SUBJECT_SAMPLE_FACTORS           	-	21610051_C	Factor:Control	RAW_FILE_NAME=21610051_C
SUBJECT_SAMPLE_FACTORS           	-	21670026_C	Factor:Control	RAW_FILE_NAME=21670026_C
SUBJECT_SAMPLE_FACTORS           	-	21703814_C	Factor:Control	RAW_FILE_NAME=21703814_C
SUBJECT_SAMPLE_FACTORS           	-	21753462_C	Factor:Control	RAW_FILE_NAME=21753462_C
SUBJECT_SAMPLE_FACTORS           	-	18862900_GA	Factor:GA	RAW_FILE_NAME=18862900_GA
SUBJECT_SAMPLE_FACTORS           	-	19533094_GA	Factor:GA	RAW_FILE_NAME=19533094_GA
SUBJECT_SAMPLE_FACTORS           	-	19558785_GA	Factor:GA	RAW_FILE_NAME=19558785_GA
SUBJECT_SAMPLE_FACTORS           	-	19568043_GA	Factor:GA	RAW_FILE_NAME=19568043_GA
SUBJECT_SAMPLE_FACTORS           	-	19762935_GA	Factor:GA	RAW_FILE_NAME=19762935_GA
SUBJECT_SAMPLE_FACTORS           	-	19785967_GA	Factor:GA	RAW_FILE_NAME=19785967_GA
SUBJECT_SAMPLE_FACTORS           	-	19844611_GA	Factor:GA	RAW_FILE_NAME=19844611_GA
SUBJECT_SAMPLE_FACTORS           	-	19864732_GA	Factor:GA	RAW_FILE_NAME=19864732_GA
SUBJECT_SAMPLE_FACTORS           	-	19885751_GA	Factor:GA	RAW_FILE_NAME=19885751_GA
SUBJECT_SAMPLE_FACTORS           	-	19929105_GA	Factor:GA	RAW_FILE_NAME=19929105_GA
SUBJECT_SAMPLE_FACTORS           	-	20249412_GA	Factor:GA	RAW_FILE_NAME=20249412_GA
SUBJECT_SAMPLE_FACTORS           	-	20249546_GA	Factor:GA	RAW_FILE_NAME=20249546_GA
SUBJECT_SAMPLE_FACTORS           	-	20308928_GA	Factor:GA	RAW_FILE_NAME=20308928_GA
SUBJECT_SAMPLE_FACTORS           	-	20310303_GA	Factor:GA	RAW_FILE_NAME=20310303_GA
SUBJECT_SAMPLE_FACTORS           	-	20312851_GA	Factor:GA	RAW_FILE_NAME=20312851_GA
SUBJECT_SAMPLE_FACTORS           	-	20334941_GA	Factor:GA	RAW_FILE_NAME=20334941_GA
SUBJECT_SAMPLE_FACTORS           	-	20407797_GA	Factor:GA	RAW_FILE_NAME=20407797_GA
SUBJECT_SAMPLE_FACTORS           	-	20488455_GA	Factor:GA	RAW_FILE_NAME=20488455_GA
SUBJECT_SAMPLE_FACTORS           	-	20492412_GA	Factor:GA	RAW_FILE_NAME=20492412_GA
SUBJECT_SAMPLE_FACTORS           	-	20525109_GA	Factor:GA	RAW_FILE_NAME=20525109_GA
SUBJECT_SAMPLE_FACTORS           	-	20537584_GA	Factor:GA	RAW_FILE_NAME=20537584_GA
SUBJECT_SAMPLE_FACTORS           	-	20624808_GA	Factor:GA	RAW_FILE_NAME=20624808_GA
SUBJECT_SAMPLE_FACTORS           	-	20639226_GA	Factor:GA	RAW_FILE_NAME=20639226_GA
SUBJECT_SAMPLE_FACTORS           	-	20735858_GA	Factor:GA	RAW_FILE_NAME=20735858_GA
SUBJECT_SAMPLE_FACTORS           	-	20749860_GA	Factor:GA	RAW_FILE_NAME=20749860_GA
SUBJECT_SAMPLE_FACTORS           	-	20774404_GA	Factor:GA	RAW_FILE_NAME=20774404_GA
SUBJECT_SAMPLE_FACTORS           	-	20777340_GA	Factor:GA	RAW_FILE_NAME=20777340_GA
SUBJECT_SAMPLE_FACTORS           	-	20780043_GA	Factor:GA	RAW_FILE_NAME=20780043_GA
SUBJECT_SAMPLE_FACTORS           	-	20788142_GA	Factor:GA	RAW_FILE_NAME=20788142_GA
SUBJECT_SAMPLE_FACTORS           	-	20830630_GA	Factor:GA	RAW_FILE_NAME=20830630_GA
SUBJECT_SAMPLE_FACTORS           	-	20854922_GA	Factor:GA	RAW_FILE_NAME=20854922_GA
SUBJECT_SAMPLE_FACTORS           	-	20855976_GA	Factor:GA	RAW_FILE_NAME=20855976_GA
SUBJECT_SAMPLE_FACTORS           	-	20876340_GA	Factor:GA	RAW_FILE_NAME=20876340_GA
SUBJECT_SAMPLE_FACTORS           	-	20927291_GA	Factor:GA	RAW_FILE_NAME=20927291_GA
SUBJECT_SAMPLE_FACTORS           	-	21033438_GA	Factor:GA	RAW_FILE_NAME=21033438_GA
SUBJECT_SAMPLE_FACTORS           	-	21294251_GA	Factor:GA	RAW_FILE_NAME=21294251_GA
SUBJECT_SAMPLE_FACTORS           	-	21349010_GA	Factor:GA	RAW_FILE_NAME=21349010_GA
SUBJECT_SAMPLE_FACTORS           	-	21355783_GA	Factor:GA	RAW_FILE_NAME=21355783_GA
SUBJECT_SAMPLE_FACTORS           	-	21368884_GA	Factor:GA	RAW_FILE_NAME=21368884_GA
SUBJECT_SAMPLE_FACTORS           	-	21371994_GA	Factor:GA	RAW_FILE_NAME=21371994_GA
SUBJECT_SAMPLE_FACTORS           	-	21437087_GA	Factor:GA	RAW_FILE_NAME=21437087_GA
SUBJECT_SAMPLE_FACTORS           	-	21443088_GA	Factor:GA	RAW_FILE_NAME=21443088_GA
SUBJECT_SAMPLE_FACTORS           	-	21444935_GA	Factor:GA	RAW_FILE_NAME=21444935_GA
SUBJECT_SAMPLE_FACTORS           	-	MOH00027337062_GA	Factor:GA	RAW_FILE_NAME=MOH00027337062_GA
SUBJECT_SAMPLE_FACTORS           	-	MOH00027340288_GA	Factor:GA	RAW_FILE_NAME=MOH00027340288_GA
SUBJECT_SAMPLE_FACTORS           	-	MOH00027354847_GA	Factor:GA	RAW_FILE_NAME=MOH00027354847_GA
SUBJECT_SAMPLE_FACTORS           	-	MOH00027361087_GA	Factor:GA	RAW_FILE_NAME=MOH00027361087_GA
#COLLECTION
CO:COLLECTION_SUMMARY            	The Institutional Review Boards at King Faisal Specialist Hospital and Research
CO:COLLECTION_SUMMARY            	Centre (KFSHRC) in Riyadh, Saudi Arabia (RAC # 2160 027) reviewed and approved
CO:COLLECTION_SUMMARY            	this study and its related procedures. DBS samples were obtained from the
CO:COLLECTION_SUMMARY            	metabolomics section laboratory, Clinical Genomics Department (CGD) in the
CO:COLLECTION_SUMMARY            	Center for Genomic Medicine (CGM) at KFSHRC as leftover material from the
CO:COLLECTION_SUMMARY            	newborn screening program. To identify predictive metabolic profiling, newborn
CO:COLLECTION_SUMMARY            	DBS samples found to have transit elevation of C5DC, showing normal C5DC level
CO:COLLECTION_SUMMARY            	in the follow-up NBS testing, were used (n=47), and DBS samples from healthy
CO:COLLECTION_SUMMARY            	controls (n=47) were included in this study for comparison purposes to predict
CO:COLLECTION_SUMMARY            	different metabolic profiling. The two groups were age- and gender-matched,
CO:COLLECTION_SUMMARY            	where non-newborn samples with ages more than 4 weeks or diagnosed with other
CO:COLLECTION_SUMMARY            	inborn errors of metabolism (IMD) were excluded from this study.
CO:COLLECTION_PROTOCOL_FILENAME  	GA_Sample Collection
CO:SAMPLE_TYPE                   	Blood (plasma)
#TREATMENT
TR:TREATMENT_SUMMARY             	None
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Metabolites were extracted from DBS using a standard procedure reported
SP:SAMPLEPREP_SUMMARY            	elsewhere (14). Briefly, one punch, a size of 3.2 mm, was collected from each
SP:SAMPLEPREP_SUMMARY            	DBS, and then metabolites were extracted by adding 250 ul (40:40:20) (methanol:
SP:SAMPLEPREP_SUMMARY            	acetonitrile: dH2O) and mixing in a plate shaker for 2 h at RT. Meanwhile,
SP:SAMPLEPREP_SUMMARY            	another set of punches from each sample was collected and pooled as QC samples,
SP:SAMPLEPREP_SUMMARY            	which were prepared to control the system's stability. After that, the extracts
SP:SAMPLEPREP_SUMMARY            	from the study and QC samples were dried using SpeedVac (Thermo Fischer, Christ,
SP:SAMPLEPREP_SUMMARY            	Germany). The dried samples were reconstituted in 90 ul using (1:1) mobile phase
SP:SAMPLEPREP_SUMMARY            	A: B (A: 0.1% Formic acid in dH2O, B: 0.1% Formic acid in 50% MeOH and ACN) and
SP:SAMPLEPREP_SUMMARY            	10 µl of internal standard mixture for metabolomics analysis.
SP:SAMPLEPREP_PROTOCOL_ID        	GA_Metabolites extraction
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	The Waters Acquity UPLC system coupled with a Xevo G2-S QTOF mass spectrometer
CH:CHROMATOGRAPHY_SUMMARY        	equipped with an electrospray ionization source (ESI) was used to analyze the
CH:CHROMATOGRAPHY_SUMMARY        	samples. The extracted metabolites were chromatographed using an ACQUITY UPLC
CH:CHROMATOGRAPHY_SUMMARY        	using XSelect (100×2.1mm, 2.5 μm) column (Waters Ltd., Elstree, UK), the
CH:CHROMATOGRAPHY_SUMMARY        	mobile phase composed of 0.1% formic acid in dH2O as solvent A, and B consists
CH:CHROMATOGRAPHY_SUMMARY        	of 0.1% formic acid in 50% ACN: MeOH. A gradient elution schedule was run as
CH:CHROMATOGRAPHY_SUMMARY        	follows: 0-16 min 95- 5% A, 16-19 min 5% A, 19-20 min 5-95% A, 20-22 min 95- 95%
CH:CHROMATOGRAPHY_SUMMARY        	A, at 300 µL/min flow rate. MS spectra were acquired under positive and
CH:CHROMATOGRAPHY_SUMMARY        	negative electrospray ionization modes (ESI+, ESI-). MS conditions were as
CH:CHROMATOGRAPHY_SUMMARY        	follows: source temperature was 150◦C, the desolvation temperature was 500◦C
CH:CHROMATOGRAPHY_SUMMARY        	(ESI+) or 140 (ESI−), the capillary voltage was 3.20 kV (ESI+) or 3 kV
CH:CHROMATOGRAPHY_SUMMARY        	(ESI−), cone voltage was 40 V, desolvation gas flow was 800.0 L/h, cone gas
CH:CHROMATOGRAPHY_SUMMARY        	flow was 50 L/h. The collision energies of low and high functions were set at
CH:CHROMATOGRAPHY_SUMMARY        	off and 10 V to 50 V, respectively, in MSE mode. The mass spectrometer was
CH:CHROMATOGRAPHY_SUMMARY        	calibrated with 100–1200 Da sodium formate in both ionization modes. The lock
CH:CHROMATOGRAPHY_SUMMARY        	mass compound, leucine-enkephalin (an external reference to the ion m/z 556.2771
CH:CHROMATOGRAPHY_SUMMARY        	in (ESI+) and 554.2615 (ESI-)), was infused continuously, switching between the
CH:CHROMATOGRAPHY_SUMMARY        	sample and the reference every 45 and 60 s for ESI+ and ESI-, respectively, for
CH:CHROMATOGRAPHY_SUMMARY        	a 0.5 s scan time, a flow rate of 10 µL/min, a cone voltage of 30 V, and
CH:CHROMATOGRAPHY_SUMMARY        	collision energy of 4 V. Data were collected in continuum mode with Masslynx™
CH:CHROMATOGRAPHY_SUMMARY        	V4.1 workstation (Waters Inc., Milford, Massachusetts, USA). The samples were
CH:CHROMATOGRAPHY_SUMMARY        	acquired randomly, and a QC sample was injected after each 10 study samples to
CH:CHROMATOGRAPHY_SUMMARY        	control the system’s variations. A mixture of several internal standards with
CH:CHROMATOGRAPHY_SUMMARY        	a final concentration of 10 µg/ml was added to each sample, serving as a
CH:CHROMATOGRAPHY_SUMMARY        	reference compound to help correct any variations that may occur during sample
CH:CHROMATOGRAPHY_SUMMARY        	preparation and analysis.
CH:METHODS_FILENAME              	GA_LCMS Metabolomics
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters Acquity UPLC XSelect HSS C18 (100 × 2.1mm, 2.5um)
CH:COLUMN_TEMPERATURE            	55
CH:FLOW_GRADIENT                 	0–16 min 95%–5% A, 16–19 min 5% A, 19–20 min 5%–95% A, and 20–22
CH:FLOW_GRADIENT                 	min, 95%– 95% A
CH:FLOW_RATE                     	300 μl/min.
CH:SOLVENT_A                     	0.1% formic acid in dH2O
CH:SOLVENT_B                     	0.1% formic acid in 50% MeOH and ACN
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Waters Xevo-G2-S
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	The DIA data were collected with a Masslynx™ V4.1 workstation in continuum
MS:MS_COMMENTS                   	mode (Waters Inc., Milford, MA, USA). The raw MS data were processed following a
MS:MS_COMMENTS                   	standard pipeline using the Progenesis QI v.3.0 software.
MS:ION_MODE                      	NEGATIVE
MS:MS_RESULTS_FILE               	ST002934_AN004813_Results.txt	UNITS:Peak Area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END