#METABOLOMICS WORKBENCH Codreags00_20240320_120618 DATATRACK_ID:4731 STUDY_ID:ST003133 ANALYSIS_ID:AN005143 PROJECT_ID:PR001946 VERSION 1 CREATED_ON March 21, 2024, 9:58 am #PROJECT PR:PROJECT_TITLE Unraveling Salivary Metabolome in Children with Eosinophilic Esophagitis: PR:PROJECT_TITLE Insights into Disease Pathogenesis and Translational Potential. PR:PROJECT_SUMMARY In this pilot study, we performed global untargeted salivary metabolomics. We PR:PROJECT_SUMMARY focused on salivary metabolomics as oral mucosa is the initial interface between PR:PROJECT_SUMMARY the triggering antigens and the host mucosal immune system,9 saliva is rich yet PR:PROJECT_SUMMARY understudied biofluid, and saliva can be collected safely, rapidly, and PR:PROJECT_SUMMARY non-invasively (compared to esophageal and plasma samples) making it uniquely PR:PROJECT_SUMMARY suited for testing pediatric populations.10 Our primary aim was to gain novel PR:PROJECT_SUMMARY insights into the upstream metabolomic alterations in pediatric EoE by profiling PR:PROJECT_SUMMARY salivary metabolome in children with EoE, and our secondary aim was to assess PR:PROJECT_SUMMARY the translational potential of salivary metabolites. PR:INSTITUTE Vanderbilt University PR:DEPARTMENT Chemistry PR:LABORATORY Center for Innovative Technology PR:LAST_NAME CODREANU PR:FIRST_NAME SIMONA PR:ADDRESS 1234 STEVENSON CENTER LANE PR:EMAIL SIMONA.CODREANU@VANDERBILT.EDU PR:PHONE 6158758422 #STUDY ST:STUDY_TITLE Unraveling Salivary Metabolome in Children with Eosinophilic Esophagitis ST:STUDY_TYPE untargeted metabolomics analysis ST:STUDY_SUMMARY In this pilot study, we performed global untargeted salivary metabolomics. We ST:STUDY_SUMMARY focused on salivary metabolomics as oral mucosa is the initial interface between ST:STUDY_SUMMARY the triggering antigens and the host mucosal immune system,9 saliva is rich yet ST:STUDY_SUMMARY understudied biofluid, and saliva can be collected safely, rapidly, and ST:STUDY_SUMMARY non-invasively (compared to esophageal and plasma samples) making it uniquely ST:STUDY_SUMMARY suited for testing pediatric populations.10 Our primary aim was to gain novel ST:STUDY_SUMMARY insights into the upstream metabolomic alterations in pediatric EoE by profiling ST:STUDY_SUMMARY salivary metabolome in children with EoE, and our secondary aim was to assess ST:STUDY_SUMMARY the translational potential of salivary metabolites. ST:INSTITUTE Vanderbilt University ST:DEPARTMENT Chemistry ST:LABORATORY Center for Innovative Technology ST:LAST_NAME CODREANU ST:FIRST_NAME SIMONA ST:ADDRESS 1234 STEVENSON CENTER LANE ST:EMAIL SIMONA.CODREANU@VANDERBILT.EDU ST:PHONE 6158758422 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 28 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN Eosinophilic esophagitis EoE SU:AGE_OR_AGE_RANGE children (6-18 years) SU:GENDER Male and female SU:HUMAN_INCLUSION_CRITERIA Children diagnosed with EoE or with symptoms suggestive of EoE and undergoing SU:HUMAN_INCLUSION_CRITERIA EGD with biopsies for clinical care were consecutively enrolled SU:HUMAN_EXCLUSION_CRITERIA Children with inflammatory bowel disease, celiac disease, connective tissue SU:HUMAN_EXCLUSION_CRITERIA disorder, prior esophageal injury or surgery, esophageal varices, use of SU:HUMAN_EXCLUSION_CRITERIA systemic steroids or antibiotics within the previous 30 days, neurodevelopmental SU:HUMAN_EXCLUSION_CRITERIA delays, or with a visible oral ulcer or gingival disease were excluded. #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 - N1 Sample source:saliva | Genotype:negative control-GERD RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_N1 SUBJECT_SAMPLE_FACTORS - N2 Sample source:saliva | Genotype:negative control-GERD RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_N2 SUBJECT_SAMPLE_FACTORS - N3 Sample source:saliva | Genotype:negative control-GERD RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_N3 SUBJECT_SAMPLE_FACTORS - N4 Sample source:saliva | Genotype:negative control-GERD RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_N4 SUBJECT_SAMPLE_FACTORS - N5 Sample source:saliva | Genotype:negative control-GERD RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_N5 SUBJECT_SAMPLE_FACTORS - P1 Sample source:saliva | Genotype:positive control- nonEoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_P1 SUBJECT_SAMPLE_FACTORS - P2 Sample source:saliva | Genotype:positive control- nonEoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_P2 SUBJECT_SAMPLE_FACTORS - P4 Sample source:saliva | Genotype:positive control- nonEoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_P4 SUBJECT_SAMPLE_FACTORS - P5 Sample source:saliva | Genotype:positive control- nonEoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_P5 SUBJECT_SAMPLE_FACTORS - A1 Sample source:saliva | Genotype:Active EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Act1 SUBJECT_SAMPLE_FACTORS - A2 Sample source:saliva | Genotype:Active EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Act2 SUBJECT_SAMPLE_FACTORS - A3 Sample source:saliva | Genotype:Active EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Act3 SUBJECT_SAMPLE_FACTORS - A4 Sample source:saliva | Genotype:Active EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Act4 SUBJECT_SAMPLE_FACTORS - A5 Sample source:saliva | Genotype:Active EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Act5 SUBJECT_SAMPLE_FACTORS - A6 Sample source:saliva | Genotype:Active EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Act6 SUBJECT_SAMPLE_FACTORS - A7 Sample source:saliva | Genotype:Active EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Act7 SUBJECT_SAMPLE_FACTORS - A8 Sample source:saliva | Genotype:Active EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Act8 SUBJECT_SAMPLE_FACTORS - A9 Sample source:saliva | Genotype:Active EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Act9 SUBJECT_SAMPLE_FACTORS - In1 Sample source:saliva | Genotype:Inactive EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Inact1 SUBJECT_SAMPLE_FACTORS - In2 Sample source:saliva | Genotype:Inactive EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Inact2 SUBJECT_SAMPLE_FACTORS - In3 Sample source:saliva | Genotype:Inactive EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Inact3* SUBJECT_SAMPLE_FACTORS - In4 Sample source:saliva | Genotype:Inactive EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Inact4 SUBJECT_SAMPLE_FACTORS - In5 Sample source:saliva | Genotype:Inactive EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Inact5 SUBJECT_SAMPLE_FACTORS - In6 Sample source:saliva | Genotype:Inactive EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Inact6 SUBJECT_SAMPLE_FACTORS - In7 Sample source:saliva | Genotype:Inactive EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Inact7 SUBJECT_SAMPLE_FACTORS - In8 Sample source:saliva | Genotype:Inactive EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Inact8 SUBJECT_SAMPLE_FACTORS - In9 Sample source:saliva | Genotype:Inactive EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Inact9 SUBJECT_SAMPLE_FACTORS - In10 Sample source:saliva | Genotype:Inactive EoE RAW_FILE_NAME(MS Sample Name)=SC_20230617_aHILICp_FMS_Locke_Inact10 #COLLECTION CO:COLLECTION_SUMMARY Saliva samples were collected immediately before the scheduled EGD. All CO:COLLECTION_SUMMARY participants were nil per os for at least 6 hours before the procedure, and the CO:COLLECTION_SUMMARY saliva samples were collected in a sterile tube (BD Biosciences, San Jose, CA) CO:COLLECTION_SUMMARY between 7:30 am and 11:30 am. After collection, the samples were maintained at CO:COLLECTION_SUMMARY 4°C and transported to the laboratory within 2 hours. In the laboratory, the CO:COLLECTION_SUMMARY saliva samples were centrifuged at 3000 rpm for 15 mins at room temperature to CO:COLLECTION_SUMMARY remove particulate debris, and the supernatant was stored in aliquots at -80°C CO:COLLECTION_SUMMARY until analysis. CO:SAMPLE_TYPE Saliva CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS sterile tube (BD Biosciences, San Jose, CA) CO:COLLECTION_TUBE_TEMP 4 #TREATMENT TR:TREATMENT_SUMMARY After collection, the samples were maintained at 4°c C and transported to the TR:TREATMENT_SUMMARY laboratory within 2 hours. In the laboratory, the saliva samples were TR:TREATMENT_SUMMARY centrifuged at 3000 rpm for 15 mins at room temperature to remove particulate TR:TREATMENT_SUMMARY debris, and the supernatant was stored in aliquots at -80°C until analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Briefly, after thawing, the samples were normalized by total protein amount (50 SP:SAMPLEPREP_SUMMARY µg) following a Bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, SP:SAMPLEPREP_SUMMARY MA). Metabolites were extracted using an ice-cold solvent of methanol/water SP:SAMPLEPREP_SUMMARY (80:20, v/v) mixture. Heavy-labeled phenylalanine-D8 and biotin-D2 was were SP:SAMPLEPREP_SUMMARY added to individual samples before protein precipitation to be able to assess SP:SAMPLEPREP_SUMMARY sample preparation reproducibility. Following overnight incubation at -80°C, SP:SAMPLEPREP_SUMMARY precipitated proteins were pelleted by centrifugation at 10,000 rpm for 10 min, SP:SAMPLEPREP_SUMMARY and metabolite extracts were then dried in vacuo and stored at -80°C. SP:SAMPLEPREP_SUMMARY Individual extracts were reconstituted in 100 µl of acetonitrile/water (80:20, SP:SAMPLEPREP_SUMMARY v/v) containing heavy-labeled carnitine-D9, tryptophan-D3, valine-D8, and SP:SAMPLEPREP_SUMMARY inosine-4N15, and centrifuged for 5 min at 10,000 rpm to remove insoluble SP:SAMPLEPREP_SUMMARY material. A pooled quality control sample (QC) was prepared by pooling equal SP:SAMPLEPREP_SUMMARY volumes of individual samples. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Following standard addition, protein precipitation was performed by adding SP:EXTRACTION_METHOD 800µL of ice-cold methanol (4x by volume). Samples were incubated at -80°C SP:EXTRACTION_METHOD overnight. Following incubation, samples were centrifuged at 10,000 rpm for 10 SP:EXTRACTION_METHOD min to eliminate proteins. The supernatants containing metabolites were dried SP:EXTRACTION_METHOD via speed-vacuum. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) CH:SOLVENT_A 90% water, 10% acetonitrile, 5mM Ammonium Formate, 0.1%FA CH:SOLVENT_B 10% water, 90% acetonitrile, 5mM Ammonium Formate, 0.1%FA CH:FLOW_GRADIENT 30 min; 95%A, 5%B CH:FLOW_RATE 0.20mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The full mass scan was acquired at 120,000 resolutions with a scan rate of 3.5 MS:MS_COMMENTS Hz and an automatic gain control (AGC) target of 1x106. A maximum ion injection MS:MS_COMMENTS time of 100 ms and MS/MS spectra were collected at 15,000 resolutions, with an MS:MS_COMMENTS AGC target of 2x105 ions and a maximum ion injection time of 100 ms All mass MS:MS_COMMENTS spectrometry data was imported, processed, normalized, and reviewed using MS:MS_COMMENTS Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK) MS:MS_RESULTS_FILE ST003133_AN005143_Results.txt UNITS:peak intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END