#METABOLOMICS WORKBENCH stuppie_20140913_9988281_mwtab.txt DATATRACK_ID:153 STUDY_ID:ST000100 ANALYSIS_ID:AN000166 PROJECT_ID:PR000092 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE Global Metabolomics of C. elegans using an augmented reference IROA design PR:PROJECT_SUMMARY - PR:INSTITUTE University of Florida PR:DEPARTMENT Biochemistry & Molecular Biology PR:LAST_NAME Edison PR:FIRST_NAME Arthur PR:ADDRESS R3-226 Academic Research Building, Department of Biochemistry & Molecular PR:ADDRESS PO Box 100245, Gainesville, Fl PR:EMAIL aedison@ufl.edu PR:PHONE (352) 392-4535 PR:FUNDING_SOURCE NIH #STUDY ST:STUDY_TITLE Global Metabolomics of C. elegans using an augmented reference IROA design ST:STUDY_TYPE heat shock ST:STUDY_SUMMARY Global Metabolomics of C. elegans using an augmented reference IROA design ST:INSTITUTE University of Florida ST:DEPARTMENT Biochemistry & Molecular Biology ST:LABORATORY Arthur Edison ST:LAST_NAME Edison ST:FIRST_NAME Arthur ST:ADDRESS R3-226 Academic Research Building, Department of Biochemistry & Molecular ST:ADDRESS PO Box 100245, Gainesville, FL 32610-0245 ST:EMAIL aedison@ufl.edu ST:PHONE - #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Caenorhabditis elegans SU:TAXONOMY_ID 6329 SU:GENOTYPE_STRAIN N2 SU:AGE_OR_AGE_RANGE Young adult SU:GENDER Hermaphrodite 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 Worm 10PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=4; BioRep=10; ExperRep=1,2; PairID=101,102; SampleID=37,38; SampleOrder=32,48 SUBJECT_SAMPLE_FACTORS Worm 11PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=4; BioRep=11; ExperRep=1,2; PairID=111,112; SampleID=41,42; SampleOrder=8,2 SUBJECT_SAMPLE_FACTORS Worm 12PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=4; BioRep=12; ExperRep=1,2; PairID=121,122; SampleID=45,46; SampleOrder=23,33 SUBJECT_SAMPLE_FACTORS Worm 1PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=1; BioRep=1; ExperRep=1,2; PairID=11,12; SampleID=1,2; SampleOrder=19,35 SUBJECT_SAMPLE_FACTORS Worm 2PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=1; BioRep=2; ExperRep=1,2; PairID=21,22; SampleID=5,6; SampleOrder=40,37 SUBJECT_SAMPLE_FACTORS Worm 3PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=1; BioRep=3; ExperRep=1,2; PairID=31,32; SampleID=9,10; SampleOrder=25,12 SUBJECT_SAMPLE_FACTORS Worm 4PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=2; BioRep=4; ExperRep=1,2; PairID=41,42; SampleID=13,14; SampleOrder=10,6 SUBJECT_SAMPLE_FACTORS Worm 5PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=2; BioRep=5; ExperRep=1,2; PairID=51,52; SampleID=17,18; SampleOrder=22,45 SUBJECT_SAMPLE_FACTORS Worm 6PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=2; BioRep=6; ExperRep=1,2; PairID=61,62; SampleID=21,22; SampleOrder=15,44 SUBJECT_SAMPLE_FACTORS Worm 7PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=3; BioRep=7; ExperRep=1,2; PairID=71,72; SampleID=25,26; SampleOrder=41,4 SUBJECT_SAMPLE_FACTORS Worm 8PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=3; BioRep=8; ExperRep=1,2; PairID=81,82; SampleID=29,30; SampleOrder=17,13 SUBJECT_SAMPLE_FACTORS Worm 9PC-5 Labelling:0.05 | Experimentalgroup:heat-shock Batch=3; BioRep=9; ExperRep=1,2; PairID=91,92; SampleID=33,34; SampleOrder=27,30 SUBJECT_SAMPLE_FACTORS Worm 10PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=4; BioRep=10; ExperRep=1,2; PairID=101,102; SampleID=39,40; SampleOrder=31,47 SUBJECT_SAMPLE_FACTORS Worm 11PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=4; BioRep=11; ExperRep=1,2; PairID=111,112; SampleID=43,44; SampleOrder=7,1 SUBJECT_SAMPLE_FACTORS Worm 12PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=4; BioRep=12; ExperRep=1,2; PairID=121,122; SampleID=47,48; SampleOrder=24,34 SUBJECT_SAMPLE_FACTORS Worm 1PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=1; BioRep=1; ExperRep=1,2; PairID=11,12; SampleID=3,4; SampleOrder=20,36 SUBJECT_SAMPLE_FACTORS Worm 2PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=1; BioRep=2; ExperRep=1,2; PairID=21,22; SampleID=7,8; SampleOrder=39,38 SUBJECT_SAMPLE_FACTORS Worm 3PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=1; BioRep=3; ExperRep=1,2; PairID=31,32; SampleID=11,12; SampleOrder=26,11 SUBJECT_SAMPLE_FACTORS Worm 4PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=2; BioRep=4; ExperRep=1,2; PairID=41,42; SampleID=15,16; SampleOrder=9,5 SUBJECT_SAMPLE_FACTORS Worm 5PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=2; BioRep=5; ExperRep=1,2; PairID=51,52; SampleID=19,20; SampleOrder=21,46 SUBJECT_SAMPLE_FACTORS Worm 6PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=2; BioRep=6; ExperRep=1,2; PairID=61,62; SampleID=23,24; SampleOrder=16,43 SUBJECT_SAMPLE_FACTORS Worm 7PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=3; BioRep=7; ExperRep=1,2; PairID=71,72; SampleID=27,28; SampleOrder=42,3 SUBJECT_SAMPLE_FACTORS Worm 8PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=3; BioRep=8; ExperRep=1,2; PairID=81,82; SampleID=31,32; SampleOrder=18,14 SUBJECT_SAMPLE_FACTORS Worm 9PHS-5 Labelling:0.05 | Experimentalgroup:no heat-shock Batch=3; BioRep=9; ExperRep=1,2; PairID=91,92; SampleID=35,36; SampleOrder=28,29 SUBJECT_SAMPLE_FACTORS Worm 10PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=4; BioRep=10; ExperRep=1,2; PairID=101,102; SampleID=37,38; SampleOrder=32,48 SUBJECT_SAMPLE_FACTORS Worm 11PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=4; BioRep=11; ExperRep=1,2; PairID=111,112; SampleID=41,42; SampleOrder=8,2 SUBJECT_SAMPLE_FACTORS Worm 12PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=4; BioRep=12; ExperRep=1,2; PairID=121,122; SampleID=45,46; SampleOrder=23,33 SUBJECT_SAMPLE_FACTORS Worm 1PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=1; BioRep=1; ExperRep=1,2; PairID=11,12; SampleID=1,2; SampleOrder=19,35 SUBJECT_SAMPLE_FACTORS Worm 2PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=1; BioRep=2; ExperRep=1,2; PairID=21,22; SampleID=5,6; SampleOrder=40,37 SUBJECT_SAMPLE_FACTORS Worm 3PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=1; BioRep=3; ExperRep=1,2; PairID=31,32; SampleID=9,10; SampleOrder=25,12 SUBJECT_SAMPLE_FACTORS Worm 4PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=2; BioRep=4; ExperRep=1,2; PairID=41,42; SampleID=13,14; SampleOrder=10,6 SUBJECT_SAMPLE_FACTORS Worm 5PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=2; BioRep=5; ExperRep=1,2; PairID=51,52; SampleID=17,18; SampleOrder=22,45 SUBJECT_SAMPLE_FACTORS Worm 6PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=2; BioRep=6; ExperRep=1,2; PairID=61,62; SampleID=21,22; SampleOrder=15,44 SUBJECT_SAMPLE_FACTORS Worm 7PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=3; BioRep=7; ExperRep=1,2; PairID=71,72; SampleID=25,26; SampleOrder=41,4 SUBJECT_SAMPLE_FACTORS Worm 8PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=3; BioRep=8; ExperRep=1,2; PairID=81,82; SampleID=29,30; SampleOrder=17,13 SUBJECT_SAMPLE_FACTORS Worm 9PC-95 Labelling:0.95 | Experimentalgroup:heat-shock Batch=3; BioRep=9; ExperRep=1,2; PairID=91,92; SampleID=33,34; SampleOrder=27,30 SUBJECT_SAMPLE_FACTORS Worm 10PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=4; BioRep=10; ExperRep=1,2; PairID=101,102; SampleID=39,40; SampleOrder=31,47 SUBJECT_SAMPLE_FACTORS Worm 11PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=4; BioRep=11; ExperRep=1,2; PairID=111,112; SampleID=43,44; SampleOrder=7,1 SUBJECT_SAMPLE_FACTORS Worm 12PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=4; BioRep=12; ExperRep=1,2; PairID=121,122; SampleID=47,58; SampleOrder=24,34 SUBJECT_SAMPLE_FACTORS Worm 1PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=1; BioRep=1; ExperRep=1,2; PairID=11,12; SampleID=3,4; SampleOrder=20,36 SUBJECT_SAMPLE_FACTORS Worm 2PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=1; BioRep=2; ExperRep=1,2; PairID=21,22; SampleID=7,8; SampleOrder=39,38 SUBJECT_SAMPLE_FACTORS Worm 3PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=1; BioRep=3; ExperRep=1,2; PairID=31,32; SampleID=11,12; SampleOrder=26,11 SUBJECT_SAMPLE_FACTORS Worm 4PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=2; BioRep=4; ExperRep=1,2; PairID=41,42; SampleID=15,16; SampleOrder=9,5 SUBJECT_SAMPLE_FACTORS Worm 5PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=2; BioRep=5; ExperRep=1,2; PairID=51,52; SampleID=19,20; SampleOrder=21,46 SUBJECT_SAMPLE_FACTORS Worm 6PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=2; BioRep=6; ExperRep=1,2; PairID=61,62; SampleID=23,24; SampleOrder=16,43 SUBJECT_SAMPLE_FACTORS Worm 7PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=3; BioRep=7; ExperRep=1,2; PairID=71,72; SampleID=27,28; SampleOrder=42,3 SUBJECT_SAMPLE_FACTORS Worm 8PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=3; BioRep=8; ExperRep=1,2; PairID=81,82; SampleID=31,32; SampleOrder=18,14 SUBJECT_SAMPLE_FACTORS Worm 9PHS-95 Labelling:0.95 | Experimentalgroup:no heat-shock Batch=3; BioRep=9; ExperRep=1,2; PairID=91,92; SampleID=35,36; SampleOrder=28,29 #COLLECTION CO:COLLECTION_SUMMARY Each flask was separated into supernatant (exometabolome) and worm pellet CO:COLLECTION_SUMMARY by centrifugation 2,000 rpm for 2 min. CO:COLLECTION_PROTOCOL_ID Centrifugation CO:COLLECTION_PROTOCOL_FILENAME Experimental_Methods.docx CO:VOLUMEORAMOUNT_COLLECTED 25,000 worms in 2.5 mL #TREATMENT TR:TREATMENT_SUMMARY Samples were treated with a 20 min heat shock at 33 °C followed by incubation TR:TREATMENT_SUMMARY 22C for 100 min. Sample shook at 250 rpm at a worm density of 10,000 worms per TR:TREATMENT_SUMMARY Samples were incubated at 22C for 120 min. Sample shook at 250 rpm at a worm TR:TREATMENT_SUMMARY of 10,000 worms per mL. TR:TREATMENT_PROTOCOL_ID Test/Control #SAMPLEPREP SP:SAMPLEPREP_SUMMARY - SP:SAMPLEPREP_PROTOCOL_ID Endometabolome SP:SAMPLEPREP_PROTOCOL_FILENAME Experimental_Methods.docx SP:SAMPLEPREP_PROTOCOL_COMMENTS The worm pellets were homogenized using a FastPrep-24 (MP Biomedicals) using SP:SAMPLEPREP_PROTOCOL_COMMENTS Matrix E tubes for 5 cycles of 60 sec in 80% methanol and then frozen at -80 SP:PROCESSING_METHOD Homogenization #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were analyzed using a mass range of m/z 70-1000 in positive and CH:CHROMATOGRAPHY_SUMMARY ionization mode, externally calibrated, using a Thermo Scientific Q-Exactive CH:CHROMATOGRAPHY_SUMMARY mass spectrometer equipped with a Dionex UltiMate 3000 RS autosampler and pump. CH:CHROMATOGRAPHY_SUMMARY ?L of each sample were injected onto an Acquity UPLC HSS T3 (2.1x100 mm, 1.8 CH:CHROMATOGRAPHY_SUMMARY column using a column temperature of 35 C and a flow rate of 300 L/min with CH:CHROMATOGRAPHY_SUMMARY A (0.1% formic acid in water (Fisher LC-MS Optima)) held at 100% A for 1 min, CH:CHROMATOGRAPHY_SUMMARY by linear increase to 80% B (0.1% formic acid in acetonitrile) over 10 min, CH:CHROMATOGRAPHY_SUMMARY constant for 2 min, followed by a return to initial conditions over 0.5 min and CH:CHROMATOGRAPHY_SUMMARY for 3 min. CH:CHROMATOGRAPHY_TYPE Reversed phase MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap CH:COLUMN_NAME Waters Acquity HSS T3 (100 x 2.1mm, 1.8um) CH:FLOW_GRADIENT A held at 100% A for 1 min, followed by linear increase to 80% B over 10 min, CH:FLOW_GRADIENT constant for 2 min, followed by a return to initial conditions over 0.5 min and CH:FLOW_GRADIENT for 3 min CH:FLOW_RATE 300 L/min CH:SAMPLE_INJECTION 3 ?L CH:SOLVENT_A 0.1% formic acid in water (Fisher LC-MS Optima) CH:SOLVENT_B 0.1% formic acid in acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Edison AN:OPERATOR_NAME Chaevien Clendinen AN:DETECTOR_TYPE Negative #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The Q-Exactive was equipped with a Heated Electrospray Ionization (HESI) source MS:MS_COMMENTS operated at a spray temperature of 350 ?C, a spray voltage of 3.5 kV, and MS:MS_COMMENTS and auxiliary gas flow rates of 50 and 10 arbitrary units, respectively. The MS:MS_COMMENTS temperature was held at 325 ?C, and the S-lens RF Level was set to 40%. The FR MS:MS_COMMENTS was set to 70,000 at m/z 200. The accuracy achieved was routinely less than 1.5 MS:CAPILLARY_TEMPERATURE 325 ?C MS:ION_SOURCE_TEMPERATURE 350 ?C MS:ION_SPRAY_VOLTAGE 3.5 kV MS:RESOLUTION_SETTING 70000 MS:MS_COMMENTS The Q-Exactive was equipped with a Heated Electrospray Ionization (HESI) source MS:MS_COMMENTS operated at a spray temperature of 350 ?C, a spray voltage of 3.5 kV, and MS:MS_COMMENTS and auxiliary gas flow rates of 50 and 10 arbitrary units, respectively. The MS:MS_COMMENTS temperature was held at 325 ?C, and the S-lens RF Level was set to 40%. The FR MS:MS_COMMENTS was set to 70,000 at m/z 200. The accuracy achieved was routinely less than 1.5 #END