#METABOLOMICS WORKBENCH Toadpup1956_20210209_120248 DATATRACK_ID:2464 STUDY_ID:ST001690 ANALYSIS_ID:AN002760 PROJECT_ID:PR001086 VERSION 1 CREATED_ON February 11, 2021, 1:00 pm #PROJECT PR:PROJECT_TITLE Parallel multi-omics in high-risk subjects for the identification of integrated PR:PROJECT_TITLE biomarker signatures of type 1 diabetes PR:PROJECT_TYPE Untargeted metabolomics: MS qualitative analysis PR:PROJECT_SUMMARY MS qualitative analysis of human blood samples to identify early-identification PR:PROJECT_SUMMARY biomarkers for type 1 diabetes PR:INSTITUTE Duke University PR:DEPARTMENT Duke Molecular Physiology Institute, School of Medicine PR:LABORATORY Metabolomics PR:LAST_NAME Bain PR:FIRST_NAME James PR:ADDRESS 300 N Duke Street, Durham, NC, 27701, USA PR:EMAIL james.bain@duke.edu PR:PHONE 919 479 2320 PR:FUNDING_SOURCE NIH, NIDDK PR:PROJECT_COMMENTS This data represents the untargeted metabolomic analysis of the samples PR:CONTRIBUTORS O. Alcazar, L.F. Hernandez, E.S. Nakayasu, C. Ansong, C.D. Nicora, C. Ansong, PR:CONTRIBUTORS M.J. Muehlbauer, J.R. Bain, C.J. Myer, S.J. Bhattacharya, P. Buchwald, and M.H. PR:CONTRIBUTORS Abdulreda #STUDY ST:STUDY_TITLE Untargeted metabolomic analysis of human blood samples via qualitative GC-MS for ST:STUDY_TITLE T1D biomarker identification ST:STUDY_TYPE Qualitative GC-MS biomarker identification ST:STUDY_SUMMARY "Blood from human subjects at high risk for T1D (and healthy controls; n=4 each) ST:STUDY_SUMMARY were subjected to parallel unlabeled proteomics, metabolomics, lipidomics, and ST:STUDY_SUMMARY transcriptomics. The integrated dataset was analyzed using Ingenuity Pathway ST:STUDY_SUMMARY Analysis (IPA) software for disturbances in the at-risk subjects compared to the ST:STUDY_SUMMARY controls. The final quadra-omics dataset contained 2292 proteins, 328 miRNAs, 75 ST:STUDY_SUMMARY metabolites, and 41 lipids that were detected in all samples. Disease/function ST:STUDY_SUMMARY enrichment analyses consistently indicated increased activation, proliferation, ST:STUDY_SUMMARY and migration of immune cells, particularly, CD4 T-lymphocytes and macrophages. ST:STUDY_SUMMARY Integrated molecular network predictions highlighted central involvement and ST:STUDY_SUMMARY activation of NF-κB, TGF-β, VEGF, arachidonic acid, and arginase, and ST:STUDY_SUMMARY inhibition of miRNA Let-7a-5p. Parallel multi-omics provided a comprehensive ST:STUDY_SUMMARY picture of disturbances in high-risk T1D subjects and helped identify an ST:STUDY_SUMMARY associated integrated biomarker signature, which could ultimately facilitate the ST:STUDY_SUMMARY classification of T1D progressors from non-progressors." ST:INSTITUTE Duke University ST:DEPARTMENT Duke Molecular Physiology Institute, School of Medicine ST:LABORATORY Metabolomics ST:LAST_NAME Bain ST:FIRST_NAME James ST:ADDRESS 300 N Duke St, Durham, NC, 27701, USA ST:EMAIL james.bain@duke.edu ST:PHONE 919 479 2320 ST:TOTAL_SUBJECTS 9 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 140365 1 Condition:High-risk T1D RAW_FILE_NAME=Miami-01 SUBJECT_SAMPLE_FACTORS 2019 0605 TL 2 Condition:Healthy control RAW_FILE_NAME=Miami-02 SUBJECT_SAMPLE_FACTORS 467191 3 Condition:New-onset (post-prandial) RAW_FILE_NAME=Miami-03 SUBJECT_SAMPLE_FACTORS 467191 small 4 Condition:New-onset (fasting) RAW_FILE_NAME=Miami-04 SUBJECT_SAMPLE_FACTORS 145569 5 Condition:High-risk T1D RAW_FILE_NAME=Miami-05 SUBJECT_SAMPLE_FACTORS 2019 0605 BH 6 Condition:Healthy control RAW_FILE_NAME=Miami-06 SUBJECT_SAMPLE_FACTORS 2019 0605 YM 7 Condition:Healthy control RAW_FILE_NAME=Miami-07 SUBJECT_SAMPLE_FACTORS 245361 8 Condition:High-risk T1D RAW_FILE_NAME=Miami-08 SUBJECT_SAMPLE_FACTORS 2019 0605 MG 9 Condition:Healthy control RAW_FILE_NAME=Miami-09 SUBJECT_SAMPLE_FACTORS Blank Ghost (blank) Condition:Process blank RAW_FILE_NAME=Ghost SUBJECT_SAMPLE_FACTORS - RT Condition:- RAW_FILE_NAME=- #COLLECTION CO:COLLECTION_SUMMARY "Blood samples (~20 mL/subject in EDTA) were collected from consented CO:COLLECTION_SUMMARY male/female subjects considered at high risk for T1D during routine visits as CO:COLLECTION_SUMMARY part of the ongoing TrialNet’s Natural History Study of the Development of CO:COLLECTION_SUMMARY Type 1 Diabetes (Pathway to Prevention Study) TN-01 study (n=4)." CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY "Subjects in the TN-01 study are tested semi-annually for the appearance of new TR:TREATMENT_SUMMARY or additional autoantibodies and are evaluated metabolically by oral glucose TR:TREATMENT_SUMMARY tolerance test (OGTT) to assess their progression toward clinical diagnosis of TR:TREATMENT_SUMMARY T1D. Samples from healthy subjects (n=4) were collected as part of another study TR:TREATMENT_SUMMARY approved by the IRB of the University of Miami (study number 11995-115). These TR:TREATMENT_SUMMARY trials are conducted in accordance with the principles of the Declaration of TR:TREATMENT_SUMMARY Helsinki and consistent with the Good Clinical Practice guidelines of the TR:TREATMENT_SUMMARY International Conference on Harmonization. The protocol for the ancillary study, TR:TREATMENT_SUMMARY under which the current multi-omics analyses were performed, was approved by TR:TREATMENT_SUMMARY TrialNet (study ID number 195) and its IRB. The four high-risk subjects in the TR:TREATMENT_SUMMARY present report were staged for their risk level according to the TrialNet TR:TREATMENT_SUMMARY staging/scoring system, which considers family history, genetic susceptibility TR:TREATMENT_SUMMARY according to haplotype (e.g., HLA-DQ/DR), the number of autoantibodies, and OGTT TR:TREATMENT_SUMMARY results as follows: low-risk (1 autoantibody and normal OGTT); moderate-risk TR:TREATMENT_SUMMARY (2–3 autoantibodies and normal OGTT); high-risk (4–5 autoantibodies and TR:TREATMENT_SUMMARY normal OGTT); and very high-risk (4–5 autoantibodies and abnormal OGTT)." #SAMPLEPREP SP:SAMPLEPREP_SUMMARY See uploaded protocol SP:SAMPLEPREP_PROTOCOL_FILENAME Toadpup1956_20210209_120248_PR_SP_PlasmaPrep_non-targeted_MBX.pdf SP:SAMPLEPREP_PROTOCOL_COMMENTS Please treat these as confidential disclosures of in-house sample prep. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 6890N CH:COLUMN_NAME Agilent DB5-MS (30m x 0.25mm, 0.25um) CH:METHODS_FILENAME Toadpup1956_20210209_120248_PR_MS_Data_acquisition_protocol.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Toadpup1956_20210209_120248_PR_MS_Data_acquisition_protocol.pdf #MS MS:INSTRUMENT_NAME Agilent 5975B MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Acquired using Agilent MassHunter Acquisition software. Feature assignment MS:MS_COMMENTS completed using AMDIS Analysis. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS minutes MS_METABOLITE_DATA_START Samples 1 2 3 4 5 6 7 8 9 Ghost (blank) Factors Condition:High-risk T1D Condition:Healthy control Condition:New-onset (post-prandial) Condition:New-onset (fasting) Condition:High-risk T1D Condition:Healthy control Condition:Healthy control Condition:High-risk T1D Condition:Healthy control Condition:Process blank Pyruvic acid 19.27 19.15 19.59 18.76 19.9 16.12 17.73 19.6 19.84 Lactic acid 24.4 24.13 24.25 24.19 25.32 24.29 23.6 24.49 24.82 12.83 2-Hydroxyisobutyric acid 13.26 13.58 12.91 Glycolic acid 16.69 15.34 16.95 17.57 17.16 15.46 16.65 3-Methyl pyruvic acid 13 11.28 Alanine 20.26 21.12 20.13 20.85 20.9 20.9 21.04 20.41 20.73 2-Ketovaline 15.6 14.05 15.48 15.65 15.46 14.26 14.84 14.33 14.76 2-Hydroxybutyric acid 17.6 18.17 18.92 18.3 19.33 19.08 18.57 18.49 19.33 P-Cresol 13.56 11.85 13.46 12.12 13.36 12.07 Leucine 17.29 17.49 17.49 17.6 17.93 17.52 18.07 17.53 16.85 3-Hydroxybutyric acid 18.74 19.55 19.25 18.93 20.34 21.96 18.92 19.94 17.72 2-Hydroxyvaleric acid 14.66 16.87 14.72 16.52 16.32 15.29 15.13 14.08 15.14 N-Methylalanine/2-Aminobutanoic acid 14.62 16.1 15.09 15.35 16.45 16.02 15.76 17.48 15.21 2-Ketoleucine/ketoisoleucine 17.22 15.55 16.87 17.03 16.97 16.78 16.22 17.02 16.96 Valine 18.53 20.28 18.78 20.15 20.39 19.47 19.59 19.83 18.98 Urea 25.05 26.03 25.25 26.2 26.22 25.5 25.6 25.73 25.4 Caprylic acid 15.42 16.97 13 15.75 15.4 15.18 16.63 16.9 14.43 Ethanolamine 14.17 13.67 14.8 15.09 15.06 15.5 16.63 17.59 14.8 Phosphoric acid 23.93 24.92 24.59 25.03 25.41 25.29 24.29 24.65 25.07 20.07 Glycerol 21.39 20.95 23.12 23.4 23.36 24.29 23.97 21.25 18.21 Isoleucine/Leucine 16.83 18.11 17.45 17.86 19.46 18.28 16.91 16.75 16.41 Proline 18.54 19.97 17.49 18.55 20.2 19.24 17.65 17.16 16.62 Glycine 18.96 18.69 19.63 19.34 20.44 20.85 19.88 20.88 20.06 Succinic acid 16.79 16.78 19.06 16.46 Maleic acid 15 15.77 16 15.8 13.3 15.97 Glyceric acid 15.72 17.13 16.36 17.89 17.09 18.28 17.08 17.32 17.57 Uracil 12.91 11.32 Fumaric acid 11.93 13.24 11.79 12.62 Serine 17.69 19.91 18.1 19.02 20.44 20.48 15.95 17.47 18.43 Pelargonic acid 11.28 13.06 12.32 13.77 12.2 13.22 Threonine 18.95 21.51 18.89 20.06 21.44 20.69 17.36 18.03 19.26 beta-Alanine 10.99 11.93 12.57 12.74 11.17 Capric acid 14.59 11.4 12.52 12.96 13.39 14.32 15.65 15.05 Aminomalonic acid 17.73 19.9 19.68 20.65 20.57 17.64 20.12 Malic acid 13.36 15.54 16.58 16.87 17.74 14.3 15.66 15.57 Threitol/Erythritol 16.11 17.92 16.24 16.98 17.39 16.81 15.71 16.49 16.69 Aspartic acid 13.3 15.96 13.56 15.24 16.14 15.35 13.01 13.78 14.41 3-Methyloxyindole 15.85 17.4 Methionine 15.81 17.41 15.46 16.73 17.85 17.05 13.66 15.84 16.04 Hydroxyprolines 16.04 16.57 16.28 16.49 19.59 16.29 13.77 15.74 16.84 Erythronic acid 14.11 16.01 14.95 16.35 16.56 16.68 15 15.66 16.23 Threonic acid 17.36 18.43 17.88 18.98 19.03 17.48 17.78 18.46 Oxoglutaric acid 15.79 17.65 18.43 17 13.88 Glutamic acid 19.33 18.53 17.58 20.63 21.72 22.02 17.96 18.45 19.52 3-Hydroxymethylglutaricacid 19.04 19.69 Phenylalanine 18.83 20.28 19.28 20.19 20.8 20.26 19.12 19.46 19.23 Lauric acid 15.65 13.94 16.6 14.72 16.19 17.14 16.29 17.45 Aldopentoses 12.92 14.46 13.41 12.77 12.15 14.67 14.89 14.96 13.87 Pyrophosphate 14.72 16.08 16.78 16.81 16.93 17.58 16.83 16.89 17.92 Asparagine 16.65 18.21 17.45 17.68 18.8 18.57 16.53 17.33 17.59 Pentitols 14.74 17.11 15.01 16.19 16.33 15.69 15.06 15.58 15.46 6-Deoxyhexose 13.78 12.78 13.13 12.54 14.44 12.79 13.69 14.08 sn-Glycero-3-phosphate 11.94 16.47 16.89 16.56 17.71 13.41 14.84 15.35 Gentisic acid 12.03 13.17 Pentonic acids 13.17 13.75 11.59 13.74 14.04 11.7 Glutamine 19.7 21.55 20.56 21.88 21.94 21.39 19.52 20.49 21.49 Phosphoethanolamine 12.9 15.11 14.77 3-Phosphoglyceric acid 16.23 16.99 14.79 Shikimic acid 15.75 16.27 16.23 15.48 17.21 16.57 14.38 17.76 Hippuric acid 13.01 14.43 3-Methylhistidine 11.81 13.32 Hypoxanthine 14.14 15.5 13.48 10.42 14.37 15.11 14.48 15.37 Citric acid/isocitric acid 19.59 21.26 20.5 21.05 22.38 22 19.63 20.99 21.15 Ornithine 18.4 18.81 18.47 18.62 19.9 19.3 19.63 19.18 18.77 Arginine/Citrulline 12.4 12.93 12.1 1,5-Anhydrosorbitol 22.01 21.46 21.09 22.36 22.51 22.43 21.13 22.22 21.44 Myristic acid 17.29 15.51 19.02 15.87 16.78 16.95 17.2 19.72 14.45 12.14 Quinic acid 13.13 14.17 13.67 15.36 15.59 Caffeine 16.51 13.54 14.84 17.48 Fructose or similar ketohexose 16.85 19.68 16.23 19 18.92 17.32 17.82 17.82 17.86 Dehydroascorbic acid 19.93 18.85 19.77 19.88 17.05 17.69 20.24 Hexose 26.45 26.76 26.49 26.71 26.74 26.63 26.56 26.57 26.99 10.45 Histidine 15.88 18.29 16.14 17.94 18.22 18.14 15.16 16.16 17.31 Lysine 18.59 20.68 19.89 20.16 21.03 20.8 19.72 20.02 20.47 Methyl palmitate 16.79 21.56 16.61 19.95 18.57 20.99 21.23 21.56 18.92 Tyrosine 19.2 20.38 19.79 19.82 20.4 20.46 19.9 20.1 19.82 Gluconic acid 18.73 19.89 19.06 19.9 20.05 20.14 19.36 19.28 21.34 Indoleacetic acid 12.82 12.78 13.23 12.66 14.29 13.6 12.82 12.31 3-Carboxy-4-methyl-5-propyl-2-furanpropanoic acid 12.9 13.21 12.37 12.5 13.73 5-Hydroxylysine 12.16 13.14 12.39 10.08 12.83 Palmitoleic acid 17.11 17.27 19.49 17.58 19.14 19.21 17.61 20.37 16.66 Palmitic acid 23.14 22.49 23.8 22.68 23.06 23.4 23.09 23.64 22.15 21.9 3-Indolepropionic acid 11.18 14.93 11.67 12.88 14.8 13.66 13.72 14.97 Myo-inositol 18.75 20.17 19.08 20.14 19.84 20.05 18.88 19.49 19.83 Uric acid 21.2 19.97 20.81 21.26 21.41 21.81 19.52 19.42 21.17 Methyl linoleate 18.78 17.57 15.42 19.41 19.58 20.59 16.27 Methyl oleate 18.22 16.56 14.23 18.02 18.37 19.14 15.47 Methyl stearate 16.57 21.52 17.98 20.46 18.72 21.84 22.02 22.58 19.53 Heptadecanoic acid/Octadecanol 13.95 12.15 14.85 13.18 13.94 14.34 14.34 15.83 10.45 3-Indolelactic acid 12.84 14.03 1-Methyltryptophan 11.85 14.44 14.26 15.72 15.77 10.55 12.52 Linoleic acid 21.79 19.53 23.05 21.15 21.53 22.52 21.8 22.78 20.46 17.04 Tryptophan 18.83 17.92 18.77 19.8 20.79 19.47 18.93 19.27 20.03 Oleic acid 22.89 20.87 23.97 21.57 22.77 23.14 23.03 23.74 20.6 14.81 Cystine 14.84 16.79 14.14 15.13 17.43 15.95 16.36 Hexose 6-phosphate 10.84 10.7 12.97 10.76 Arachidonic acid 16.26 17.53 17.91 18.78 17.61 19.14 18.2 18.21 18.34 16.18 Arachidic acid/ 1-Heneicosanol 12.4 13.99 13.97 13.65 13.24 13.63 13.79 11.87 5-Methoxytryptamine/Serotonin 16.89 16.66 17.07 MG 0:0/16:0/0:0 17.14 17.8 18.26 18.04 18.02 17.93 17.93 17.8 18.04 15 Docosahexaenoic acid 14.36 14.48 12.27 15.77 15.31 14.28 14.26 Cellobiose or similar disaccharide 16.26 18.86 14.97 17.75 17.57 19.8 19.19 16.9 18.05 MG 0:0/18:0/0:0 20.52 21.08 21.55 21.22 21.04 21.08 21.15 20.95 21.17 18.44 MG 18:1(9Z)/0:0/0:0 12.24 13.06 13.54 13.02 14.07 14.07 13.6 15.34 13.87 12.38 Maltose or similar disaccharide 12.46 16.64 11.93 15.22 14.67 17.79 17.06 13.67 15.6 gamma-Tocopherol 15.03 16.35 16.77 15.35 15.43 16.06 15.51 15.74 16.79 AMP 12.58 10.36 alpha-Tocopherol 17.85 19.6 19.24 19.65 18.84 19.65 19.62 19.76 19.35 Cholesterol 24.06 25.82 25.32 25.25 24.86 25.67 25.75 25.68 25.22 Campesterol 11.63 13.16 13.07 16.7 12.74 Sitosterol 12.89 13.88 15.81 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention time Pyruvic acid 6.599 Lactic acid 6.753 2-Hydroxyisobutyric acid 6.813 Glycolic acid 7.005 3-Methyl pyruvic acid 7.384 Alanine 7.403 2-Ketovaline 7.49 2-Hydroxybutyric acid 7.749 P-Cresol 8.203 Leucine 8.233 3-Hydroxybutyric acid 8.258 2-Hydroxyvaleric acid 8.307 N-Methylalanine/2-Aminobutanoic acid 8.438 2-Ketoleucine/ketoisoleucine 9.032 Valine 9.073 Urea 9.526 Caprylic acid 9.808 Ethanolamine 9.816 Phosphoric acid 9.877 Glycerol 9.885 Isoleucine/Leucine 10.18 Proline 10.264 Glycine 10.374 Succinic acid 10.478 Maleic acid 10.551 Glyceric acid 10.663 Uracil 10.798 Fumaric acid 10.977 Serine 11.079 Pelargonic acid 11.145 Threonine 11.419 beta-Alanine 12.012 Capric acid 12.419 Aminomalonic acid 12.532 Malic acid 12.744 Threitol/Erythritol 12.971 Aspartic acid 13.154 3-Methyloxyindole 13.16 Methionine 13.168 Hydroxyprolines 13.236 Erythronic acid 13.408 Threonic acid 13.607 Oxoglutaric acid 13.83 Glutamic acid 14.362 3-Hydroxymethylglutaricacid 14.362 Phenylalanine 14.479 Lauric acid 14.79 Aldopentoses 14.806 Pyrophosphate 14.801 Asparagine 14.932 Pentitols 15.471 6-Deoxyhexose 15.547 sn-Glycero-3-phosphate 15.932 Gentisic acid 15.945 Pentonic acids 15.953 Glutamine 16.104 Phosphoethanolamine 16.191 3-Phosphoglyceric acid 16.416 Shikimic acid 16.417 Hippuric acid 16.457 3-Methylhistidine 16.467 Hypoxanthine 16.482 Citric acid/isocitric acid 16.569 Ornithine 16.575 Arginine/Citrulline 16.655 1,5-Anhydrosorbitol 16.931 Myristic acid 16.948 Quinic acid 16.975 Caffeine 17.026 Fructose or similar ketohexose 17.178 Dehydroascorbic acid 17.189 Hexose 17.359 Histidine 17.642 Lysine 17.671 Methyl palmitate 17.769 Tyrosine 17.845 Gluconic acid 17.985 Indoleacetic acid 18.106 3-Carboxy-4-methyl-5-propyl-2-furanpropanoic acid 18.239 5-Hydroxylysine 18.531 Palmitoleic acid 18.727 Palmitic acid 18.923 3-Indolepropionic acid 19.277 Myo-inositol 19.297 Uric acid 19.363 Methyl linoleate 19.414 Methyl oleate 19.473 Methyl stearate 19.706 Heptadecanoic acid/Octadecanol 19.847 3-Indolelactic acid 20.084 1-Methyltryptophan 20.095 Linoleic acid 20.457 Tryptophan 20.465 Oleic acid 20.508 Cystine 21.133 Hexose 6-phosphate 21.303 Arachidonic acid 21.817 Arachidic acid/ 1-Heneicosanol 22.408 5-Methoxytryptamine/Serotonin 22.539 MG 0:0/16:0/0:0 23.267 Docosahexaenoic acid 23.343 Cellobiose or similar disaccharide 24.624 MG 0:0/18:0/0:0 24.711 MG 18:1(9Z)/0:0/0:0 24.798 Maltose or similar disaccharide 24.825 gamma-Tocopherol 26.521 AMP 26.889 alpha-Tocopherol 27.466 Cholesterol 27.612 Campesterol 28.313 Sitosterol 28.945 METABOLITES_END #END