#METABOLOMICS WORKBENCH KGDowling_20250319_132846 DATATRACK_ID:5747 STUDY_ID:ST003810 ANALYSIS_ID:AN006264 PROJECT_ID:PR002383 VERSION 1 CREATED_ON March 25, 2025, 10:34 am #PROJECT PR:PROJECT_TITLE Longitudinal multi-omics analysis of cord blood and childhood serum in Autism PR:PROJECT_TITLE Spectrum Disorder PR:PROJECT_TYPE Untargeted Metabolomics study PR:PROJECT_SUMMARY Metabolomic analysis was performed on cord blood plasma from 22 children PR:PROJECT_SUMMARY diagnosed with Autism before age 5, and 44 neurotypical controls from the Cork PR:PROJECT_SUMMARY Baseline Birth Cohort. LC-MS analysis was performed using an ACQUITY ultra PR:PROJECT_SUMMARY performance liquid chromatography (Waters Corp, Milford, MA) coupled with a PR:PROJECT_SUMMARY Synapt G2-S quadrupole time-of-flight (UPLC-Q-TOF) mass spectrometer (Waters PR:PROJECT_SUMMARY Corp, Wilmslow, UK). MSE data were uploaded onto Progenesis QI 2.4 software PR:PROJECT_SUMMARY (Nonlinear Dynamics, Newcastle, U.K.) for peak picking and alignment. Data were PR:PROJECT_SUMMARY normalized to ‘All Compounds’ and the coefficient of variation (CV) was PR:PROJECT_SUMMARY computed for each compound across all QC runs (n=8) to evaluate repeatability of PR:PROJECT_SUMMARY measurement. Compounds with > 20% CV within the QCs were removed. Any compound PR:PROJECT_SUMMARY which had >30% missing values across the entire sample set or >30% in either PR:PROJECT_SUMMARY subgroup (case or control) was removed. Remaining features (429 negative mode, PR:PROJECT_SUMMARY 1353 positive mode) were log2 transformed and normalised on the median values. PR:PROJECT_SUMMARY Mann-Whitney U tests for significant differences between cases and controls were PR:PROJECT_SUMMARY then carried out and corrected for false discovery rate (FDR) using the PR:PROJECT_SUMMARY Benjamini-Hochberg procedure. Altered metabolites (p < 0.05) were uploaded to PR:PROJECT_SUMMARY Metaboanalyst (v6.0) to identify key metabolic pathways implicated. We PR:PROJECT_SUMMARY identified 32 metabolites as significantly altered between cases and controls PR:PROJECT_SUMMARY (see A. Noone et al. 2025, and supplementary data). PR:INSTITUTE University College Cork PR:DEPARTMENT Anatomy & Neuroscience PR:LABORATORY Dr Jane English Lab PR:LAST_NAME English PR:FIRST_NAME Jane PR:ADDRESS University College Cork PR:EMAIL jane.english@ucc.ie PR:PHONE 0879915949 PR:FUNDING_SOURCE Health Research board Ireland PR:PUBLICATIONS A.Noone et al. Molecular Psychiatry 2025 PR:CONTRIBUTORS Kirsten Dowling and Jane English #STUDY ST:STUDY_TITLE Longitudinal multi-omics analysis of cord blood and childhood serum in Autism ST:STUDY_TITLE Spectrum Disorder (Positive mode) ST:STUDY_TYPE LC MS/MS quantitative analysis ST:STUDY_SUMMARY Untargeted Metabolomic analysis was performed on cord blood plasma from 22 ST:STUDY_SUMMARY children diagnosed with Autism before age 5, and 44 neurotypical controls from ST:STUDY_SUMMARY the Cork Baseline Birth Cohort. ST:INSTITUTE University College Cork ST:DEPARTMENT Anatomy & Neuroscience ST:LABORATORY Dr Jane English Lab ST:LAST_NAME English ST:FIRST_NAME Jane ST:ADDRESS University College Cork ST:EMAIL jane.english@ucc.ie ST:PHONE 0879915949 ST:NUM_GROUPS 22 Autism cases vs 44 Neurotypical Controls ST:TOTAL_SUBJECTS 66 cord blood plasma samples ST:STUDY_COMMENTS Autism case vs control study design ST:PUBLICATIONS A.Noone et al. Molecular Psychiatry 2025 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE Birth SU:GENDER Male and female #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 - 210312_01 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_01 SUBJECT_SAMPLE_FACTORS - 210312_02 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_02 SUBJECT_SAMPLE_FACTORS - 210312_03 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_03 SUBJECT_SAMPLE_FACTORS - 210312_04 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_04 SUBJECT_SAMPLE_FACTORS - 210312_05 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_05 SUBJECT_SAMPLE_FACTORS - 210312_06 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_06 SUBJECT_SAMPLE_FACTORS - 210312_08 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_08 SUBJECT_SAMPLE_FACTORS - 210312_09 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_09 SUBJECT_SAMPLE_FACTORS - 210312_10 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_10 SUBJECT_SAMPLE_FACTORS - 210312_11 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_11 SUBJECT_SAMPLE_FACTORS - 210312_12 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_12 SUBJECT_SAMPLE_FACTORS - 210312_13 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_13 SUBJECT_SAMPLE_FACTORS - 210312_14 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_14 SUBJECT_SAMPLE_FACTORS - 210312_15 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_15 SUBJECT_SAMPLE_FACTORS - 210312_16 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_16 SUBJECT_SAMPLE_FACTORS - 210312_17 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_17 SUBJECT_SAMPLE_FACTORS - 210312_18 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_18 SUBJECT_SAMPLE_FACTORS - 210312_19 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_19 SUBJECT_SAMPLE_FACTORS - 210312_20 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_20 SUBJECT_SAMPLE_FACTORS - 210312_21 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_21 SUBJECT_SAMPLE_FACTORS - 210312_22 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_22 SUBJECT_SAMPLE_FACTORS - 210312_23 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_23 SUBJECT_SAMPLE_FACTORS - 210312_24 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_24 SUBJECT_SAMPLE_FACTORS - 210312_25 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_25 SUBJECT_SAMPLE_FACTORS - 210312_26 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_26 SUBJECT_SAMPLE_FACTORS - 210312_27 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_27 SUBJECT_SAMPLE_FACTORS - 210312_28 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_28 SUBJECT_SAMPLE_FACTORS - 210312_29 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_29 SUBJECT_SAMPLE_FACTORS - 210312_30 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_30 SUBJECT_SAMPLE_FACTORS - 210312_31 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_31 SUBJECT_SAMPLE_FACTORS - 210312_32 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_32 SUBJECT_SAMPLE_FACTORS - 210312_33 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_33 SUBJECT_SAMPLE_FACTORS - 210312_34 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_34 SUBJECT_SAMPLE_FACTORS - 210312_35 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_35 SUBJECT_SAMPLE_FACTORS - 210312_36 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_36 SUBJECT_SAMPLE_FACTORS - 210312_37 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_37 SUBJECT_SAMPLE_FACTORS - 210312_38 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_38 SUBJECT_SAMPLE_FACTORS - 210312_39 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_39 SUBJECT_SAMPLE_FACTORS - 210312_40 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_40 SUBJECT_SAMPLE_FACTORS - 210312_41 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_41 SUBJECT_SAMPLE_FACTORS - 210312_42 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_42 SUBJECT_SAMPLE_FACTORS - 210312_43 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_43 SUBJECT_SAMPLE_FACTORS - 210312_44 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_44 SUBJECT_SAMPLE_FACTORS - 210312_45 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_45 SUBJECT_SAMPLE_FACTORS - 210312_46 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_46 SUBJECT_SAMPLE_FACTORS - 210312_47 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_47 SUBJECT_SAMPLE_FACTORS - 210312_48 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_48 SUBJECT_SAMPLE_FACTORS - 210312_49 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_49 SUBJECT_SAMPLE_FACTORS - 210312_50 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_50 SUBJECT_SAMPLE_FACTORS - 210312_51 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_51 SUBJECT_SAMPLE_FACTORS - 210312_52 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_52 SUBJECT_SAMPLE_FACTORS - 210312_53 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_53 SUBJECT_SAMPLE_FACTORS - 210312_54 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_54 SUBJECT_SAMPLE_FACTORS - 210312_55 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_55 SUBJECT_SAMPLE_FACTORS - 210312_56 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_56 SUBJECT_SAMPLE_FACTORS - 210312_57 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_57 SUBJECT_SAMPLE_FACTORS - 210312_58 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_58 SUBJECT_SAMPLE_FACTORS - 210312_59 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_59 SUBJECT_SAMPLE_FACTORS - 210312_60 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_60 SUBJECT_SAMPLE_FACTORS - 210312_61 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_61 SUBJECT_SAMPLE_FACTORS - 210312_62 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_62 SUBJECT_SAMPLE_FACTORS - 210312_63 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_63 SUBJECT_SAMPLE_FACTORS - 210312_64 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_64 SUBJECT_SAMPLE_FACTORS - 210312_65 Case_ Control Status:Case | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_65 SUBJECT_SAMPLE_FACTORS - 210312_66 Case_ Control Status:Control | Sample source:Cord blood_plasma RAW_FILE_NAME=210312_66 #COLLECTION CO:COLLECTION_SUMMARY Cord blood EDTA plasma aliquots were retrieved from the INFANT ISO accredited CO:COLLECTION_SUMMARY biobanking facilities and stored at −80 °C until analysed. Blood for the CO:COLLECTION_SUMMARY later childhood assessment was collected in BD Vacutainer serum tubes. Following CO:COLLECTION_SUMMARY centrifugation, 250µl aliquots (Wilmut) were stored in −80 °C long-term CO:COLLECTION_SUMMARY storage. Local standard operating protocols were followed in all handling and CO:COLLECTION_SUMMARY storage of human bio-samples. CO:COLLECTION_PROTOCOL_FILENAME Noone_et_al_Cord_Blood_Metabolomics_Protocol.pdf CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Case control study. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For metabolomics profiling in cord blood, we used 50µl of cord blood plasma, to SP:SAMPLEPREP_SUMMARY which 150µl of cold acetonitrile was added, vortexed for 1 min, and chilled for SP:SAMPLEPREP_SUMMARY 1 hour at -20˚C. The samples were centrifuged for 15 min at 15,000 rpm; SP:SAMPLEPREP_SUMMARY supernatant was transferred to a clean tube and dried in a vacuum centrifuge at SP:SAMPLEPREP_SUMMARY 30˚C. The sample was reconstituted in 100µl of 50:50 acetonitrile:water with SP:SAMPLEPREP_SUMMARY 0.1% formic acid and transferred to glass autosampler vials for injection onto a SP:SAMPLEPREP_SUMMARY Waters ACQUITY-SYNAPT G2-S LC-MS/MS system. LC-MS/MS methodology for SP:SAMPLEPREP_SUMMARY metabolomics analysis is described in Morillon et al. SP:SAMPLEPREP_PROTOCOL_FILENAME Noone_et_al_Cord_Blood_Metabolomics_Protocol.pdf SP:PROCESSING_STORAGE_CONDITIONS -20℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY LC-MS analysis was performed using an ACQUITY ultra performance liquid CH:CHROMATOGRAPHY_SUMMARY chromatography (Waters Corp, Milford, MA) coupled with a Synapt G2-S quadrupole CH:CHROMATOGRAPHY_SUMMARY time-of-flight (UPLC-Q-TOF) mass spectrometer (Waters Corp, Wilmslow, UK) CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (150 x 1.7mm,2.1um) CH:SOLVENT_A 0.1% formic acid in water CH:SOLVENT_B 0.1% formic acid in acetonitrile CH:FLOW_GRADIENT 15min CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 65 °C CH:TARGET_SAMPLE_TEMPERATURE 7oC CH:RANDOMIZATION_ORDER yes CH:CHROMATOGRAPHY_COMMENTS pooled QC injected every tenth injection throughout the analysis. #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Noone_et_al_Cord_Blood_Metabolomics_Protocol.pdf #MS MS:INSTRUMENT_NAME Waters Synapt G2 S QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS MSE data were uploaded onto Progenesis QI 2.4 software (Nonlinear Dynamics, MS:MS_COMMENTS Newcastle, U.K.) for peak picking and alignment. Data were normalized to ‘All MS:MS_COMMENTS Compounds’ and the coefficient of variation (CV) was computed for each MS:MS_COMMENTS compound across all QC runs (n=8) to evaluate repeatability of measurement. MS:MS_COMMENTS Compounds with > 20% CV within the QCs were removed. Any compound which had >30% MS:MS_COMMENTS missing values across the entire sample set or >30% in either subgroup (case or MS:MS_COMMENTS control) was removed. Remaining features (429 negative mode, 1353 positive mode) MS:MS_COMMENTS were log2 transformed and normalised on the median values. Mann-Whitney U tests MS:MS_COMMENTS for significant differences between cases and controls were then carried out and MS:MS_COMMENTS corrected for false discovery rate (FDR) using the Benjamini-Hochberg procedure. MS:MS_COMMENTS Altered metabolites (p < 0.05) were uploaded to Metaboanalyst (v6.0) to identify MS:MS_COMMENTS key metabolic pathways implicated. Lastly, Spearman’s correlation coefficient MS:MS_COMMENTS was employed to assess for correlation between steroid metabolites. MS:MS_RESULTS_FILE ST003810_AN006264_Results.txt UNITS:m/z Has m/z:Yes Has RT:Yes RT units:Minutes #END