#METABOLOMICS WORKBENCH denagarcia_20170511_123109 DATATRACK_ID:903 STUDY_ID:ST001386 ANALYSIS_ID:AN002314 PROJECT_ID:PR000950 VERSION 1 CREATED_ON June 1, 2020, 3:21 pm #PROJECT PR:PROJECT_TITLE TEDDY Metabolomics Study PR:PROJECT_SUMMARY The quantification of primary metabolites in human plasma from TEDDY PR:PROJECT_SUMMARY case-control subjects. The TEDDY study aims to generate a comprehensive PR:PROJECT_SUMMARY understanding of how metabolic signatures in blood are affected in prediabetic PR:PROJECT_SUMMARY autoimmunity and diabetes. PR:INSTITUTE University of California, Davis PR:LABORATORY WCMC Metabolomics Core PR:LAST_NAME Fiehn PR:FIRST_NAME Oliver PR:ADDRESS 451 Health Sci Drive, Davis, CA 95616 PR:EMAIL ofiehn@ucdavis.edu PR:PHONE (530) 754-8258 #STUDY ST:STUDY_TITLE TEDDY Metabolomics Study ST:STUDY_SUMMARY Primary metabolites were quantified in human plasma from the 1:3 matched TEDDY ST:STUDY_SUMMARY case-control subjects. Information on the nested case-control study design can ST:STUDY_SUMMARY found in: Biomarker discovery study design for type 1 diabetes in The ST:STUDY_SUMMARY Environmental Determinants of Diabetes in the Young (TEDDY) study. Lee HS, ST:STUDY_SUMMARY Burkhardt B, McLeod W, Smith S, Eberhard C, Lynch K, Hadley D, Rewers M, Simell ST:STUDY_SUMMARY O, She JX, Hagopian W, Lernmark A, Akolkar B, Ziegler AG, Krischer J, and the ST:STUDY_SUMMARY TEDDY Study Group. Diabetes/Metabolism Research and Reviews. Epub 2013 December ST:STUDY_SUMMARY 15. doi: 10.1002/dmrr.2510 (PubMed ID: 24339168). Primary metabolites were ST:STUDY_SUMMARY extracted from 30 µl plasma aliquots by adding 1 ml of a carefully degassed -20 ST:STUDY_SUMMARY °C cold isopropanol/acetonitrile/water mixture (3:3:2, v/v/v) for 5 min at 4 ST:STUDY_SUMMARY °C which simultaneously precipitates proteins. After centrifugation, half of ST:STUDY_SUMMARY the extract was dried and cleaned up from triglycerides by a 50% acetonitrile ST:STUDY_SUMMARY mixture. After drying, internal standards were added as C08-C30 fatty acid ST:STUDY_SUMMARY methyl esters in chloroform as retention index markers (Kind et. al, 2009). ST:STUDY_SUMMARY Primary metabolites were derivatized by methoximation and trimethylsilylation. ST:STUDY_SUMMARY Primary metabolites were analyzed by cold injection/automatic liner exchange gas ST:STUDY_SUMMARY chromatography time-of- flight mass spectrometry (CIS/ALEX GC-TOF MS) (Fiehn et. ST:STUDY_SUMMARY al, 2008). In order to limit buildup of involatile material in the GC system and ST:STUDY_SUMMARY to prevent any carry over, an automatic liner exchange with multi-baffled liners ST:STUDY_SUMMARY and cold injection procedures was used instead of classic hot injections into ST:STUDY_SUMMARY standard s/sl liners. Multi-baffled inert glass liners were used because classic ST:STUDY_SUMMARY glass wool liners might hamper derivatization of amino groups for amino acid ST:STUDY_SUMMARY analysis. Robotic derivatization was further used to control reaction times (Ji ST:STUDY_SUMMARY et. al, 2011); and GC-columns were employed with integrated 10 meter guard ST:STUDY_SUMMARY columns, which could cut multiple times in 10 cm increments whenever quality ST:STUDY_SUMMARY control samples determined out-of-control situations. A temperature of 280 °C ST:STUDY_SUMMARY was determined to be the optimum transfer line temperature at which even ST:STUDY_SUMMARY higher-boiling compounds did not show tailing effects and at which the electron ST:STUDY_SUMMARY ionization filaments could still be operated at their optimal temperature of 250 ST:STUDY_SUMMARY °C and -70 eV. The mass spectrometer was operated using daily mass calibration ST:STUDY_SUMMARY auto-tuning using FC43 (perfluorotributyl-amine) and acquired 17 spectra per ST:STUDY_SUMMARY second and 1850-1950 V detector voltage. This high spectral acquisition rate was ST:STUDY_SUMMARY necessary to obtain enough data for mass spectral deconvolution of co-eluting ST:STUDY_SUMMARY compounds. Under these conditions, the system was around 10-times more sensitive ST:STUDY_SUMMARY than classic quadrupole GC-MS instruments and also clearly outperformed ST:STUDY_SUMMARY GC-triple quadrupole mass spectrometers. For select compounds, even lower limits ST:STUDY_SUMMARY of detection were achieved than for optimized MRM conditions in UPLC-QTRAP MS ST:STUDY_SUMMARY analysis. Around 144 unique metabolites were detectable in blood plasma (Fiehn & ST:STUDY_SUMMARY Kind, 2007); in addition to 221 unidentified compounds that were captured in the ST:STUDY_SUMMARY BinBase database system and hence, were comparable across studies. References: ST:STUDY_SUMMARY 1) Kind T, Wohlgemuth G, Lee DY, Lu Y, Palazoglu M, Shahbaz S, Fiehn O: ST:STUDY_SUMMARY FiehnLib: mass spectral and retention index libraries for metabolomics based on ST:STUDY_SUMMARY quadrupole and time-of-flight gas chromatography/mass spectrometry. Anal Chem ST:STUDY_SUMMARY 2009, 81(24):10038-10048. 2) Fiehn O, Wohlgemuth G, Scholz M, Kind T, Lee DY, Lu ST:STUDY_SUMMARY Y, Moon S, Nikolau B: Quality control for plant metabolomics: reporting ST:STUDY_SUMMARY MSI-compliant studies. Plant J 2008, 53(4):691-704. 3) Ji Y, Hebbring S, Zhu H, ST:STUDY_SUMMARY Jenkins GD, Biernacka J, Snyder K, Drews M, Fiehn O, Zeng Z, Schaid D et al: ST:STUDY_SUMMARY Glycine and a glycine dehydrogenase (GLDC) SNP as citalopram/escitalopram ST:STUDY_SUMMARY response biomarkers in depression: pharmacometabolomics-informed ST:STUDY_SUMMARY pharmacogenomics. Clin Pharmacol Ther 2011, 89(1):97-104. 4) Fiehn O, Kind T: ST:STUDY_SUMMARY Metabolite profiling in blood plasma. Methods Mol Biol 2007, 358:3-17. An ST:STUDY_SUMMARY explanation of the study design variables are explained in detail in a data ST:STUDY_SUMMARY dictionary provided in the raw data download section. ST:INSTITUTE University of South Florida ST:LABORATORY UC Davis Genome Center ST:LAST_NAME Krischer ST:FIRST_NAME Jeffrey ST:ADDRESS 3650 Spectrum Blvd, Tampa, FL 33612 ST:EMAIL TEDDYDataPlatform@epi.usf.edu ST:PHONE 8133969512 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 951627 8223325 Sex:Male | cc:6 ia_case_ind_1=1; ia_endptage_1=273; ia_case=1; ia_case_ind_2=-; ia_endptage_2=-; ia_control=-; t1d_case_ind_1=-; t1d_endptage_1=-; t1d_case=-; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_8223325.cdf.gz; Visit=9; Age in Days=273 SUBJECT_SAMPLE_FACTORS 886647 8333208 Sex:Male | cc:6 ia_case_ind_1=-; ia_endptage_1=-; ia_case=-; ia_case_ind_2=-; ia_endptage_2=-; ia_control=-; t1d_case_ind_1=-; t1d_endptage_1=-; t1d_case=-; t1d_case_ind_2=21; t1d_endptage_2=725; 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Visit=18; Age in Days=559 SUBJECT_SAMPLE_FACTORS 507620 1243302 Sex:Male | cc:6 ia_case_ind_1=30; ia_endptage_1=355; ia_case=1; ia_case_ind_2=-; ia_endptage_2=-; ia_control=-; t1d_case_ind_1=10; t1d_endptage_1=1028; t1d_case=1; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_1243302.cdf.gz; Visit=21; Age in Days=629 SUBJECT_SAMPLE_FACTORS 507620 5673652 Sex:Male | cc:6 ia_case_ind_1=30; ia_endptage_1=355; ia_case=1; ia_case_ind_2=-; ia_endptage_2=-; ia_control=-; t1d_case_ind_1=10; t1d_endptage_1=1028; t1d_case=1; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_5673652.cdf.gz; Visit=27; Age in Days=820 SUBJECT_SAMPLE_FACTORS 507620 7983283 Sex:Male | cc:6 ia_case_ind_1=30; ia_endptage_1=355; ia_case=1; ia_case_ind_2=-; ia_endptage_2=-; ia_control=-; t1d_case_ind_1=10; t1d_endptage_1=1028; t1d_case=1; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_7983283.cdf.gz; Visit=30; Age in Days=916 SUBJECT_SAMPLE_FACTORS 406926 8316506 Sex:Female | cc:6 ia_case_ind_1=-; 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Visit=6; Age in Days=204 SUBJECT_SAMPLE_FACTORS 750986 6212465 Sex:Male | cc:2 ia_case_ind_1=416; ia_endptage_1=468; ia_case=1; ia_case_ind_2=-; ia_endptage_2=-; ia_control=-; t1d_case_ind_1=-; t1d_endptage_1=-; t1d_case=-; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_6212465.cdf.gz; Visit=9; Age in Days=279 SUBJECT_SAMPLE_FACTORS 750986 5364011 Sex:Male | cc:2 ia_case_ind_1=416; ia_endptage_1=468; ia_case=1; ia_case_ind_2=-; ia_endptage_2=-; ia_control=-; t1d_case_ind_1=-; t1d_endptage_1=-; t1d_case=-; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_5364011.cdf.gz; Visit=15; Age in Days=468 SUBJECT_SAMPLE_FACTORS 750986 9816630 Sex:Male | cc:2 ia_case_ind_1=416; ia_endptage_1=468; ia_case=1; ia_case_ind_2=-; ia_endptage_2=-; ia_control=-; t1d_case_ind_1=-; t1d_endptage_1=-; t1d_case=-; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_9816630.cdf.gz; Visit=12; Age in Days=372 SUBJECT_SAMPLE_FACTORS 465287 1863789 Sex:Female | cc:2 ia_case_ind_1=417; 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t1d_case=-; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_7588066.cdf.gz; Visit=3; Age in Days=125 SUBJECT_SAMPLE_FACTORS 991314 8375415 Sex:Female | cc:3 ia_case_ind_1=-; ia_endptage_1=-; ia_case=-; ia_case_ind_2=295; ia_endptage_2=588; ia_control=1; t1d_case_ind_1=-; t1d_endptage_1=-; t1d_case=-; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_8375415.cdf.gz; Visit=12; Age in Days=384 SUBJECT_SAMPLE_FACTORS 991314 6794949 Sex:Female | cc:3 ia_case_ind_1=-; ia_endptage_1=-; ia_case=-; ia_case_ind_2=295; ia_endptage_2=588; ia_control=1; t1d_case_ind_1=-; t1d_endptage_1=-; t1d_case=-; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_6794949.cdf.gz; Visit=18; Age in Days=588 SUBJECT_SAMPLE_FACTORS 866995 2843796 Sex:Female | cc:5 ia_case_ind_1=-; ia_endptage_1=-; ia_case=-; ia_case_ind_2=367; ia_endptage_2=307; ia_control=1; t1d_case_ind_1=-; t1d_endptage_1=-; t1d_case=-; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_2843796.cdf.gz; Visit=9; Age in Days=279 SUBJECT_SAMPLE_FACTORS 866995 2034059 Sex:Female | cc:5 ia_case_ind_1=-; ia_endptage_1=-; ia_case=-; ia_case_ind_2=367; ia_endptage_2=307; ia_control=1; t1d_case_ind_1=-; t1d_endptage_1=-; t1d_case=-; t1d_case_ind_2=-; t1d_endptage_2=-; t1d_control=-; RAW_FILE_NAME=TEDDY_GCTOF_2034059.cdf.gz; Visit=6; Age in Days=209 #COLLECTION CO:COLLECTION_SUMMARY Plasma was obtained from human whole blood every 3 months up until 48 months; CO:COLLECTION_SUMMARY after 48 months, plasma was collected every 3 to 6 months depending on antibody CO:COLLECTION_SUMMARY status. CO:SAMPLE_TYPE Plasma #TREATMENT TR:TREATMENT_SUMMARY n/a #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Blood plasma or serum is extracted following the protocols published e.g. in SP:SAMPLEPREP_SUMMARY Fiehn et al. PLoS ONE (2010) e15234. 30 ?L aliquots are extracted by 1 mL of SP:SAMPLEPREP_SUMMARY degassed acetonitrile : isopropanol : water (3:3:2,v/v/v ) at –20°C, SP:SAMPLEPREP_SUMMARY centrifuged and decanted with subsequent evaporation of the solvent to complete SP:SAMPLEPREP_SUMMARY dryness. A clean-up step with acetonitrile/water (1:1) removes membrane lipids SP:SAMPLEPREP_SUMMARY and triglycerides. The cleaned extract is aliquoted into two equal portions and SP:SAMPLEPREP_SUMMARY the supernatant is dried down again. Internal standards C08-C30 FAMEs are added SP:SAMPLEPREP_SUMMARY and the sample is derivatized by methoxyamine hydrochloride in pyridine and SP:SAMPLEPREP_SUMMARY subsequently by N-methyl-N-trimethylsilyltrifluoroacetamide for SP:SAMPLEPREP_SUMMARY trimethylsilylation of acidic protons. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Leco Pegasus IV GC CH:COLUMN_NAME Restek Rtx-5Sil MS (30 x 0.25mm, 0.25um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Leco Pegasus IV TOF MS:INSTRUMENT_TYPE GC-TOF MS:MS_TYPE EI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS See protocol. MS:MS_RESULTS_FILE ST001386_AN002314_Results.txt UNITS:peak heights Has m/z:Yes Has RT:Yes RT units:Minutes #END