#METABOLOMICS WORKBENCH abeerotb12_20230529_001611 DATATRACK_ID:4051 STUDY_ID:ST002750 ANALYSIS_ID:AN004462 PROJECT_ID:PR001713
VERSION             	1
CREATED_ON             	June 20, 2023, 6:32 pm
#PROJECT
PR:PROJECT_TITLE                 	A Comprehensive Metabolomics Profile for Newborns with Maple syrup urine disease
PR:PROJECT_TYPE                  	Untargeted LCMS
PR:PROJECT_SUMMARY               	Investigation of MSUD’s distinctive profile in newborn MSUD patients using
PR:PROJECT_SUMMARY               	untargeted metabolomics
PR:INSTITUTE                     	King Saud University
PR:DEPARTMENT                    	Biochemistry
PR:LABORATORY                    	Biochemistry
PR:LAST_NAME                     	Alotaibi
PR:FIRST_NAME                    	Abeer
PR:ADDRESS                       	2808
PR:EMAIL                         	abeerotb12@gmail.com
PR:PHONE                         	966551933703
#STUDY
ST:STUDY_TITLE                   	A Comprehensive Metabolomics Profile for Newborns with Maple syrup urine disease
ST:STUDY_TYPE                    	Untargeted LCMS
ST:STUDY_SUMMARY                 	Background: Maple syrup urine disease (MSUD) is a genetic inherited disorder
ST:STUDY_SUMMARY                 	caused by a defect in the branched-chain alpha-ketoacid dehydrogenase (BCKAD)
ST:STUDY_SUMMARY                 	complex function. This complex usually breaks down three amino acids: leucine,
ST:STUDY_SUMMARY                 	isoleucine, and valine. Therefore, abnormal activity in this process, can impact
ST:STUDY_SUMMARY                 	important bodily functions and lead to metabolic dysregulation related to the
ST:STUDY_SUMMARY                 	disease complications. A wide range of studied endogenous metabolites and
ST:STUDY_SUMMARY                 	dysregulated biomarkers and pathways provide a huge core support for the
ST:STUDY_SUMMARY                 	treatment and follow-up of newborn MSUD patients. Objectives: In this study, we
ST:STUDY_SUMMARY                 	aim to investigate MSUD’s distinctive profile in newborn MSUD patients using
ST:STUDY_SUMMARY                 	untargeted metabolomics to contribute to the growing knowledge surrounding MSUD
ST:STUDY_SUMMARY                 	and pathways involved for improving patient outcomes. Methods: In this study,
ST:STUDY_SUMMARY                 	untargeted metabolomics analyses via liquid chromatography–mass spectrometry
ST:STUDY_SUMMARY                 	was used to investigate metabolic changes in dry blood spot (DBS) of 22 MSUD
ST:STUDY_SUMMARY                 	newborns and 22 healthy newborns. Results: The metabolomics results revealed
ST:STUDY_SUMMARY                 	1040 significantly dysregulated metabolites, where 303 and 737 were up- and
ST:STUDY_SUMMARY                 	down-regulated, respectively. 480 metabolites were annotated and 210 were
ST:STUDY_SUMMARY                 	identified as endogenous metabolites. The study identified potential biomarkers
ST:STUDY_SUMMARY                 	for MSUD such as L-Alloisoleucine and Methionine sulfoxide were upregulated in
ST:STUDY_SUMMARY                 	MSUD newborn compared to healthy newborns, while LysoPI was downregulated in
ST:STUDY_SUMMARY                 	MSUD newborns. In addition, the most affected pathways in MSUD Newborns were
ST:STUDY_SUMMARY                 	ascorbate and aldarate, Pentose and glucuronate interconversions and pyrimidine
ST:STUDY_SUMMARY                 	metabolism. Conclusion: Our results demonstrate metabolomics as a noninvasive
ST:STUDY_SUMMARY                 	strategy to understand the pathophysiology of the disease and is a promising
ST:STUDY_SUMMARY                 	tool to evaluate the potential biomarkers in the early diagnosis of newborn
ST:STUDY_SUMMARY                 	MSUD. Future studies are needed to correlate these dysregulated metabolites with
ST:STUDY_SUMMARY                 	defective mechanisms.
ST:INSTITUTE                     	King Saud University
ST:DEPARTMENT                    	Biochemistry
ST:LABORATORY                    	Biochemistry
ST:LAST_NAME                     	Alotaibi
ST:FIRST_NAME                    	Abeer
ST:ADDRESS                       	2808
ST:EMAIL                         	abeerotb12@gmail.com
ST:PHONE                         	966551933703
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:AGE_OR_AGE_RANGE              	</=14 days
SU:GENDER                        	Male and female
SU:HUMAN_INCLUSION_CRITERIA      	</=14 days, MSUD newborn DBS, Males and females
SU:HUMAN_EXCLUSION_CRITERIA      	>14 days and any IEM's newborn other than MSUD, unknown gender
#FACTORS
#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           	-	21903669	Sex:F | Age_Days:9 | sample_type:control	RAW_FILE_NAME=21903669.mzML
SUBJECT_SAMPLE_FACTORS           	-	21915585	Sex:F | Age_Days:6 | sample_type:control	RAW_FILE_NAME=21915585.mzML
SUBJECT_SAMPLE_FACTORS           	-	21061334	Sex:F | Age_Days:7 | sample_type:control	RAW_FILE_NAME=21061334.mzML
SUBJECT_SAMPLE_FACTORS           	-	21916609	Sex:F | Age_Days:6 | sample_type:control	RAW_FILE_NAME=21916609.mzML
SUBJECT_SAMPLE_FACTORS           	-	21903696	Sex:F | Age_Days:6 | sample_type:control	RAW_FILE_NAME=21903696.mzML
SUBJECT_SAMPLE_FACTORS           	-	21916593	Sex:F | Age_Days:9 | sample_type:control	RAW_FILE_NAME=21916593.mzML
SUBJECT_SAMPLE_FACTORS           	-	20990701	Sex:F | Age_Days:2 | sample_type:control	RAW_FILE_NAME=20990701.mzML
SUBJECT_SAMPLE_FACTORS           	-	21756797	Sex:F | Age_Days:6 | sample_type:control	RAW_FILE_NAME=21756797.mzML
SUBJECT_SAMPLE_FACTORS           	-	21756371	Sex:F | Age_Days:5 | sample_type:control	RAW_FILE_NAME=21756371.mzML
SUBJECT_SAMPLE_FACTORS           	-	154124	Sex:M | Age_Days:16 | sample_type:control	RAW_FILE_NAME=154124.mzML
SUBJECT_SAMPLE_FACTORS           	-	20882794	Sex:M | Age_Days:10 | sample_type:control	RAW_FILE_NAME=20882794.mzML
SUBJECT_SAMPLE_FACTORS           	-	20972893	Sex:M | Age_Days:5 | sample_type:control	RAW_FILE_NAME=20972893.mzML
SUBJECT_SAMPLE_FACTORS           	-	21051744	Sex:M | Age_Days:4 | sample_type:control	RAW_FILE_NAME=21051744.mzML
SUBJECT_SAMPLE_FACTORS           	-	21915503	Sex:M | Age_Days:5 | sample_type:control	RAW_FILE_NAME=21915503.mzML
SUBJECT_SAMPLE_FACTORS           	-	21751941	Sex:M | Age_Days:11 | sample_type:control	RAW_FILE_NAME=21751941.mzML
SUBJECT_SAMPLE_FACTORS           	-	21915628	Sex:M | Age_Days:5 | sample_type:control	RAW_FILE_NAME=21915628.mzML
SUBJECT_SAMPLE_FACTORS           	-	21812002	Sex:M | Age_Days:7 | sample_type:control	RAW_FILE_NAME=21812002.mzML
SUBJECT_SAMPLE_FACTORS           	-	17981165	Sex:M | Age_Days:8 | sample_type:control	RAW_FILE_NAME=17981165.mzML
SUBJECT_SAMPLE_FACTORS           	-	20830861	Sex:M | Age_Days:10 | sample_type:control	RAW_FILE_NAME=20830861.mzML
SUBJECT_SAMPLE_FACTORS           	-	21918555	Sex:M | Age_Days:13 | sample_type:control	RAW_FILE_NAME=21918555.mzML
SUBJECT_SAMPLE_FACTORS           	-	21798030	Sex:M | Age_Days:9 | sample_type:control	RAW_FILE_NAME=21798030.mzML
SUBJECT_SAMPLE_FACTORS           	-	19839244	Sex:M | Age_Days:9 | sample_type:control	RAW_FILE_NAME=19839244.mzML
SUBJECT_SAMPLE_FACTORS           	-	21309465	Sex:F | Age_Days:8 | sample_type:MSUD	RAW_FILE_NAME=21309465.mzML
SUBJECT_SAMPLE_FACTORS           	-	18821169	Sex:F | Age_Days:10 | sample_type:MSUD	RAW_FILE_NAME=18821169.mzML
SUBJECT_SAMPLE_FACTORS           	-	17943253	Sex:F | Age_Days:8 | sample_type:MSUD	RAW_FILE_NAME=17943253.mzML
SUBJECT_SAMPLE_FACTORS           	-	21359910	Sex:F | Age_Days:12 | sample_type:MSUD	RAW_FILE_NAME=21359910.mzML
SUBJECT_SAMPLE_FACTORS           	-	21323632	Sex:F | Age_Days:3 | sample_type:MSUD	RAW_FILE_NAME=21323632.mzML
SUBJECT_SAMPLE_FACTORS           	-	21722019	Sex:F | Age_Days:10 | sample_type:MSUD	RAW_FILE_NAME=21722019.mzML
SUBJECT_SAMPLE_FACTORS           	-	21383148	Sex:F | Age_Days:5 | sample_type:MSUD	RAW_FILE_NAME=21383148.mzML
SUBJECT_SAMPLE_FACTORS           	-	21432000	Sex:F | Age_Days:12 | sample_type:MSUD	RAW_FILE_NAME=21432000.mzML
SUBJECT_SAMPLE_FACTORS           	-	21432578	Sex:F | Age_Days:5 | sample_type:MSUD	RAW_FILE_NAME=21432578.mzML
SUBJECT_SAMPLE_FACTORS           	-	20741664	Sex:M | Age_Days:9 | sample_type:MSUD	RAW_FILE_NAME=20741664.mzML
SUBJECT_SAMPLE_FACTORS           	-	20649047	Sex:M | Age_Days:7 | sample_type:MSUD	RAW_FILE_NAME=20649047.mzML
SUBJECT_SAMPLE_FACTORS           	-	20990589	Sex:M | Age_Days:3 | sample_type:MSUD	RAW_FILE_NAME=20990589.mzML
SUBJECT_SAMPLE_FACTORS           	-	21288205	Sex:M | Age_Days:3 | sample_type:MSUD	RAW_FILE_NAME=21288205.mzML
SUBJECT_SAMPLE_FACTORS           	-	21439094	Sex:M | Age_Days:4 | sample_type:MSUD	RAW_FILE_NAME=21439094.mzML
SUBJECT_SAMPLE_FACTORS           	-	21439216	Sex:M | Age_Days:10 | sample_type:MSUD	RAW_FILE_NAME=21439216.mzML
SUBJECT_SAMPLE_FACTORS           	-	17762490	Sex:M | Age_Days:11 | sample_type:MSUD	RAW_FILE_NAME=17762490.mzML
SUBJECT_SAMPLE_FACTORS           	-	18097694	Sex:M | Age_Days:9 | sample_type:MSUD	RAW_FILE_NAME=18097694.mzML
SUBJECT_SAMPLE_FACTORS           	-	21483280	Sex:M | Age_Days:6 | sample_type:MSUD	RAW_FILE_NAME=21483280.mzML
SUBJECT_SAMPLE_FACTORS           	-	18725627	Sex:M | Age_Days:6 | sample_type:MSUD	RAW_FILE_NAME=18725627.mzML
SUBJECT_SAMPLE_FACTORS           	-	21221237	Sex:M | Age_Days:12 | sample_type:MSUD	RAW_FILE_NAME=21221237.mzML
SUBJECT_SAMPLE_FACTORS           	-	21917361	Sex:M | Age_Days:5 | sample_type:MSUD	RAW_FILE_NAME=21917361.mzML
SUBJECT_SAMPLE_FACTORS           	-	27192849	Sex:M | Age_Days:10 | sample_type:MSUD	RAW_FILE_NAME=27192849.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Forty-four DBS samples were collected form biochemically and genetically
CO:COLLECTION_SUMMARY            	confirmed MSUD newborns (n=22) at King Faisal Specialist Hospital and Research
CO:COLLECTION_SUMMARY            	Center (KFSHRC) and healthy controls (n=22). These healthy individuals were
CO:COLLECTION_SUMMARY            	almost age-sex matched with MSUD's group (Female 53%). Samples from newborn
CO:COLLECTION_SUMMARY            	patients and controls elder than 14 days were excluded from this study, as well
CO:COLLECTION_SUMMARY            	as any IEM other than MSUD excluded.
CO:COLLECTION_PROTOCOL_FILENAME  	MSUD_biological_samples.docx
CO:SAMPLE_TYPE                   	Dry Blood Spot
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	No treatment used
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Briefly, one punch, a size 3.3 mm, DBS from MSUD newborn and healthy controls
SP:SAMPLEPREP_SUMMARY            	and were distributed in 96 V-shaped plate wells then immersed with 250 μL of
SP:SAMPLEPREP_SUMMARY            	Extraction solvent (Water: MeOH: ACN) [20:40:40%]. The samples were vortexed in
SP:SAMPLEPREP_SUMMARY            	thermomixer (Eppendrof, Germany) at 600 rpm, 25 ˚C, for 2hrs. The samples were
SP:SAMPLEPREP_SUMMARY            	spun down at 16.000 rpm, 4 ˚C, for 10 min. The supernatants were transferred
SP:SAMPLEPREP_SUMMARY            	into new 96 V-shaped plate and the punches discards, and then the samples were
SP:SAMPLEPREP_SUMMARY            	dried in a vacuum concentrator SpeedVac (Christ, City, Germany). Dry residue was
SP:SAMPLEPREP_SUMMARY            	re-dissolved in 100 μL of methanol/water with a ratio (1:1) earlier of LC-MS
SP:SAMPLEPREP_SUMMARY            	analysis.
SP:SAMPLEPREP_PROTOCOL_FILENAME  	Metabolites_extraction.docx
SP:EXTRACT_STORAGE               	Room temperature
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity UPLC
CH:COLUMN_NAME                   	Waters XSelect CSH C18 (100 x 2.1mm 2.5um)
CH:SOLVENT_A                     	0.1% formic acid: dH2O
CH:SOLVENT_B                     	0.1% formic acid: 50% MeOH and ACN
CH:FLOW_GRADIENT                 	95–5% A [0–16 min], 5% A [16–19 min], 5–95% A [19–20 min], and
CH:FLOW_GRADIENT                 	95–95% A [20–22 min].
CH:FLOW_RATE                     	300 μL/min
CH:COLUMN_TEMPERATURE            	55
CH:METHODS_FILENAME              	LC_MS_Metabolomics_MSUD.docx
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Waters Xevo-G2-S
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Same as for POSITIVE mode
MS:MS_RESULTS_FILE               	ST002750_AN004462_Results.txt	UNITS:Peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END