#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