#METABOLOMICS WORKBENCH csclendinen_20140925_9133211_mwtab.txt DATATRACK_ID:155 TEXT OUTPUT STUDY_ID:ST000103 ANALYSIS_ID:AN000171 PROJECT_ID:PR000095 VERSION 1 CREATED_ON 10-02-2015 #PROJECT PR:PROJECT_TITLE 13C Metabolomics using INADEQUATE PR:PROJECT_SUMMARY Demonstrate a method using high-sensitivity NMR to identify an unknown PR:PROJECT_SUMMARY metabolite from a fraction isolated from an IROA LC-MS experiment. PR:INSTITUTE University of Florida PR:DEPARTMENT Department of Biochemistry and Molecular Biology / SECIM PR:LABORATORY Edison PR:LAST_NAME Edison PR:FIRST_NAME Arthur PR:EMAIL aedison@ufl.edu PR:PHONE 352-392-4535 PR:ADDRESS - #STUDY ST:STUDY_TITLE 2D-INADEQUATE NMR study of C. elegans metabolome ST:STUDY_TYPE Heats Shock Worms ST:STUDY_SUMMARY 2D INADEQUATE NMR datasets were collected from the endo and exo metabolome of ST:STUDY_SUMMARY heat shocked and control C. elegans ST:INSTITUTE University of Florida ST:DEPARTMENT Department of Biochemistry and Molecular Biology / SECIM ST:LABORATORY Arthur Edison ST:LAST_NAME Chaevien ST:FIRST_NAME Clendinen ST:ADDRESS R3-226 Academic Research Building, Department of Biochemistry & Molecular ST:ADDRESS Biology, PO Box 100245, Gainesville, FL 32610-0245 ST:EMAIL csclendinen@ufl.edu ST:PHONE 352-392-4535 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 4 #SUBJECT SU:SUBJECT_TYPE ANIMAL SU:SUBJECT_SPECIES Caenorhabditis elegans SU:TAXONOMY_ID 6239 SU:GENOTYPE_STRAIN Wild Type (N2) SU:GENDER Hermaphrodites SU:SPECIES_GROUP Invertebrate #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Ceanorhabditis elegans Control_1 Incubation Temperature:22 C SUBJECT_SAMPLE_FACTORS Ceanorhabditis elegans Control_2 Incubation Temperature:22 C SUBJECT_SAMPLE_FACTORS Ceanorhabditis elegans Control_3 Incubation Temperature:22 C SUBJECT_SAMPLE_FACTORS Ceanorhabditis elegans Control_4 Incubation Temperature:22 C SUBJECT_SAMPLE_FACTORS Ceanorhabditis elegans HS_1 Incubation Temperature:33 C SUBJECT_SAMPLE_FACTORS Ceanorhabditis elegans HS_2 Incubation Temperature:33 C SUBJECT_SAMPLE_FACTORS Ceanorhabditis elegans HS_3 Incubation Temperature:33 C SUBJECT_SAMPLE_FACTORS Ceanorhabditis elegans HS_4 Incubation Temperature:33 C #COLLECTION CO:COLLECTION_SUMMARY Supernatant (exometabolome) and worm pellet (endometabolome) were collected from CO:COLLECTION_SUMMARY each flask via centrifugation. The supernatant was filtered using a 20 ?m nylon CO:COLLECTION_SUMMARY syringe filter and lyophilized. The worm pellets were homogenized using a CO:COLLECTION_SUMMARY Biospec Mini-Beadbeater 8 in 80% methanol and dried. CO:SAMPLE_TYPE Whole worm extract CO:COLLECTION_LOCATION University of Florida CO:STORAGE_CONDITIONS -80 celsius #TREATMENT TR:TREATMENT_SUMMARY Two generations of wild-type C. elegans (N2) were fed 99% labeled e. coli. Upon TR:TREATMENT_SUMMARY reaching the young adult stage, 4 replicates of 1 million worms each were TR:TREATMENT_SUMMARY designated as the experimental population and were treated to a 30 minute heat TR:TREATMENT_SUMMARY shock by incubating at 33 °C in the absence of food. After 30 minutes of heat TR:TREATMENT_SUMMARY shock, the experimental populations were incubated at room temperature (22 °C) TR:TREATMENT_SUMMARY for an additional 1.5 hours. In addition 4 additional replicates of 1 million TR:TREATMENT_SUMMARY worms each were designated as the control population and was incubated at room TR:TREATMENT_SUMMARY temperature (22 °C) 2 hours in the absence of food. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Exometabolome samples were resuspended in 50 ul of Dueterium Oxide (D2O) and the SP:SAMPLEPREP_SUMMARY Endometabolome were resuspended in 50 ul of Deuterated Methanol (MeOD). SP:PROCESSING_METHOD Lyophilization SP:EXTRACT_STORAGE -80 celsius SP:SAMPLE_RESUSPENSION 50 microliters of D2O or MeOD #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:LABORATORY_NAME Arthur Edison AN:ANALYSIS_TYPE NMR AN:SOFTWARE_VERSION VNMRJ AN:OPERATOR_NAME Chaevien Clendinen #NMR NM:INSTRUMENT_TYPE Varian NM:NMR_EXPERIMENT_TYPE 2D-INADEQUATE NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_PROBE 1.5 mm HTS probe NM:NMR_SOLVENT D2O and MeOD NM:NMR_TUBE_SIZE 1.5 mm NM:PULSE_SEQUENCE INADEQUATEAD NM:PULSE_WIDTH 15.6 NM:POWER_LEVEL 37 NM:RECEIVER_GAIN 60 NM:TEMPERATURE 23 NM:NUMBER_OF_SCANS 4 NM:DUMMY_SCANS 32 NM:ACQUISITION_TIME 0.067 NM:RELAXATION_DELAY 3 sec NM:SPECTRAL_WIDTH 30487.8 Hz NM:NUM_DATA_POINTS_ACQUIRED 4096 NM:REAL_DATA_POINTS 2048 NM:ZERO_FILLING 2x NM:APODIZATION Lorentz-to-Gaussian NM:BASELINE_CORRECTION_METHOD POLY NM:INSTRUMENT_NAME Agilent VNMRS-600 #NMR_BINNED_DATA NMR_BINNED_DATA:UNITS Peak area NMR_BINNED_DATA_START Bin range(ppm) Control_1 Control_2 Control_3 Control_4 HS_1 HS_2 HS_3 HS_4 NMR_BINNED_DATA_END #END