#METABOLOMICS WORKBENCH cwhitaker_20170516_121517 DATATRACK_ID:909 STUDY_ID:ST000610 ANALYSIS_ID:AN000935 PROJECT_ID:PR000446 VERSION 1 CREATED_ON May 16, 2017, 1:55 pm #PROJECT PR:PROJECT_TITLE CHEAR Urine Reference Material PR:PROJECT_TYPE Broad spectrum, reverse phase LCMS metabolomics PR:PROJECT_SUMMARY CHEAR UrineRef_20160721 was provided by Emory University. The material was PR:PROJECT_SUMMARY prepared and analyzed using the Metabolomics Reverse Phase Broad Spectrum PR:PROJECT_SUMMARY analysis on a Synapt G2-Si system. Six samples were injected of the sample PR:PROJECT_SUMMARY reference material that were prepared in replicate. PR:INSTITUTE RTI International PR:DEPARTMENT APC PR:LAST_NAME Fennell PR:FIRST_NAME Timothy PR:ADDRESS 3040 E Cornwallis Rd, Durham, North Carolina, 27709, USA PR:EMAIL fennell@rti.org PR:PHONE 9194852781 PR:FUNDING_SOURCE U2CES026544 #STUDY ST:STUDY_TITLE CHEAR Urine Reference Material ST:STUDY_TYPE Broad spectrum, reverse phase LCMS metabolomics ST:STUDY_SUMMARY CHEAR UrineRef_20160721 was provided by Emory University. The material was ST:STUDY_SUMMARY prepared and analyzed using the Metabolomics Reverse Phase Broad Spectrum ST:STUDY_SUMMARY analysis on a Synapt G2-Si system. Six samples were injected of the sample ST:STUDY_SUMMARY reference material that were prepared in replicate. ST:INSTITUTE RTI International ST:DEPARTMENT ACP ST:LAST_NAME Fennell ST:FIRST_NAME Timothy ST:ADDRESS 3040 E Cornwallis Rd, Durham, North Carolina, 27709, USA ST:EMAIL fennell@rti.org ST:PHONE 9194852781 ST:NUM_GROUPS 1 ST:TOTAL_SUBJECTS 6 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:SUBJECT_COMMENTS Urine #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - UP_A_03_7 Matrix:Urine .raw file name (POS)=UP_A_03_7_POS.raw .raw file name (NEG)=UP_A_03_7_NEG.raw Emory ID=UrineRef_20160721 SUBJECT_SAMPLE_FACTORS - UP_A_03_8 Matrix:Urine .raw file name (POS)=UP_A_03_8_POS.raw .raw file name (NEG)=UP_A_03_8_NEG.raw Emory ID=UrineRef_20160721 SUBJECT_SAMPLE_FACTORS - UP_A_03_9 Matrix:Urine .raw file name (POS)=UP_A_03_9_POS.raw .raw file name (NEG)=UP_A_03_9_NEG.raw Emory ID=UrineRef_20160721 SUBJECT_SAMPLE_FACTORS - UP_A_03_10 Matrix:Urine .raw file name (POS)=UP_A_03_10_POS.raw .raw file name (NEG)=UP_A_03_10_NEG.raw Emory ID=UrineRef_20160721 SUBJECT_SAMPLE_FACTORS - UP_A_03_11 Matrix:Urine .raw file name (POS)=UP_A_03_11_POS.raw .raw file name (NEG)=UP_A_03_11_NEG.raw Emory ID=UrineRef_20160721 SUBJECT_SAMPLE_FACTORS - UP_A_03_12 Matrix:Urine .raw file name (POS)=UP_A_03_12_POS.raw .raw file name (NEG)=UP_A_03_12_NEG.raw Emory ID=UrineRef_20160721 #COLLECTION CO:COLLECTION_SUMMARY N/A CO:SAMPLE_TYPE Urine CO:STORAGE_CONDITIONS -80˚C #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Urine: Parent and Sub-Aliquots The CHEAR UrineRef_20160721 (500 mL) was removed SP:SAMPLEPREP_SUMMARY from -80°C storage and thawed by storing it at 4°C overnight. The following SP:SAMPLEPREP_SUMMARY day, the thawed urine was inspected to ensure there was no ice remaining and the SP:SAMPLEPREP_SUMMARY bulk urine was then mixed in the 4°C room by repeatedly inverting the SP:SAMPLEPREP_SUMMARY container. Parent aliquots were quickly prepared in the 4°C room using the SP:SAMPLEPREP_SUMMARY Drummond pipet aid and serological pipet. 42 mL urine aliquots were transferred SP:SAMPLEPREP_SUMMARY to 50 mL tubes in 4°C room and capped immediately and placed on ice. If needed, SP:SAMPLEPREP_SUMMARY the bulk urine was mixed in between aliquots. The parent aliquots were labeled SP:SAMPLEPREP_SUMMARY appropriately. Sub-aliquots were prepared on ice at the bench. The parent SP:SAMPLEPREP_SUMMARY aliquots were mixed by inverting the tube thoroughly before and in between SP:SAMPLEPREP_SUMMARY aliquots as needed. 2.5 mL urine aliquots were transferred to 5 mL cryovials and SP:SAMPLEPREP_SUMMARY capped immediately and stored on ice until sample splitting was competed. The SP:SAMPLEPREP_SUMMARY sub aliquots were labeled appropriately. Sub-aliquots were stored at -80°C. SP:SAMPLEPREP_SUMMARY Urine Aliquots for LCMS platforms: The sub-aliquot UP_A_03 was used to prepare SP:SAMPLEPREP_SUMMARY aliquots for various LCMS platforms. UP_A_03 was thawed on ice for 30 – 60 min SP:SAMPLEPREP_SUMMARY and vortexed aliquot briefly on a vortexer, followed by centrifugation at 4 °C SP:SAMPLEPREP_SUMMARY for 2 minutes at 16,000 rcf. Six aliquots of 60 µL volumes were prepared for SP:SAMPLEPREP_SUMMARY Reverse Phase analysis from UP_A_03. Aliquots were stored at -80 °C until SP:SAMPLEPREP_SUMMARY analysis. Sample preparation for Reverse Phase: The 6 urine aliquots were thawed SP:SAMPLEPREP_SUMMARY on ice for 30–60 min, and then 50 µL of each sample was transferred into a SP:SAMPLEPREP_SUMMARY new pre-labeled Lo-Bind Eppendorf tube. A volume of 400 µL methanol containing SP:SAMPLEPREP_SUMMARY 0.025 mg/mL tryptophan-d5 as the internal standard was added to extract SP:SAMPLEPREP_SUMMARY metabolites by vortex for 2 mins at 5000 rpm. Sample solution was centrifuged at SP:SAMPLEPREP_SUMMARY 16,000 rcf for 4 min at room temperature to precipitate proteins. A volume of SP:SAMPLEPREP_SUMMARY 350 µL of supernatant was transferred to pre-labeled 2.0 mL Lo-bind tubes and SP:SAMPLEPREP_SUMMARY dried down using a SpeedVac system (LabConco CentriVap) at room temperature. A SP:SAMPLEPREP_SUMMARY volume of 100 µL of Water/Methanol solution (95:5) was added to reconstitute SP:SAMPLEPREP_SUMMARY each sample. Samples were then thoroughly mixed by vortex for 10 mins at 5000 SP:SAMPLEPREP_SUMMARY rpm and then centrifuged at 16,000 rcf for 4 min at room temperature. The SP:SAMPLEPREP_SUMMARY supernatants were transferred to pre-labeled autosampler vials before analysis. SP:SAMPLEPREP_SUMMARY LC-MS analysis: The prepared urine samples were analyzed by an LC-MS system SP:SAMPLEPREP_SUMMARY comprised of a Waters Acquity UPLC and a Synapt G2Si ESI-Q-TOF mass SP:SAMPLEPREP_SUMMARY spectrometer. Chromatographic separation was accomplished on an Acquity HSST3 SP:SAMPLEPREP_SUMMARY C18 column (2.1 X 100 mm, 1.8 µm) at 50 °C using a gradient elution comprised SP:SAMPLEPREP_SUMMARY by mobile phase A: 0.1% formic acid-water (v/v), and mobile phase B: 0.1% formic SP:SAMPLEPREP_SUMMARY acid-methanol (v/v), using a flow rate of 0.400 mL/min. The injection volume was SP:SAMPLEPREP_SUMMARY 5 µL. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Reversed-Phase Gradient Seperation CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity HSS T3 (100 x 2.1mm, 1.8um) CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 50 °C CH:SOLVENT_A Water with 0.1% formic acid CH:SOLVENT_B Methanol with 0.1% formic acid CH:COLUMN_PRESSURE 6,000-10,000 psi CH:INJECTION_TEMPERATURE 8 °C CH:INTERNAL_STANDARD L-Tryptophan-d5 CH:ANALYTICAL_TIME 22 min CH:WEAK_WASH_SOLVENT_NAME 10% MeOH CH:WEAK_WASH_VOLUME 1000 µL CH:STRONG_WASH_SOLVENT_NAME 80% MeOH CH:STRONG_WASH_VOLUME 1000 µL CH:TARGET_SAMPLE_TEMPERATURE 8 °C CH:SAMPLE_LOOP_SIZE 10 µL CH:SAMPLE_SYRINGE_SIZE 100 µL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:CAPILLARY_VOLTAGE 1.0 kV MS:COLLISION_ENERGY 4 MS:FRAGMENTATION_METHOD CID, Mse MS:HELIUM_FLOW 180 MS:IONIZATION ES- MS:MASS_ACCURACY 10 ppm MS:SOURCE_TEMPERATURE 110 °C MS:DATAFORMAT Continuum MS:DESOLVATION_GAS_FLOW 1000 L/Hr MS:DESOLVATION_TEMPERATURE 400 °C MS:RESOLUTION_SETTING 20000 MS:SCAN_RANGE_MOVERZ 50-1000 m/z MS:SCANNING_CYCLE 0.3 s MS:TUBE_LENS_VOLTAGE 75 MS:MS_RESULTS_FILE ST000610_AN000935_Results.txt UNITS:Normalized Abundance #END