#METABOLOMICS WORKBENCH cwhitaker_20170516_085101 DATATRACK_ID:908 STUDY_ID:ST000609 ANALYSIS_ID:AN000933 PROJECT_ID:PR000446 VERSION 1 CREATED_ON May 26, 2017, 11:50 am #PROJECT PR:PROJECT_TITLE CHEAR Plasma Reference Material PR:PROJECT_TYPE Broad spectrum, reverse phase LCMS metabolomics PR:PROJECT_SUMMARY CHEAR PlasmaRef_20160726 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 Plasma Reference Material ST:STUDY_TYPE Broad spectrum, reverse phase LCMS metabolomics ST:STUDY_SUMMARY CHEAR PlasmaRef_20160726 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 cwhitaker@rti.org ST:PHONE 9194852781 ST:NUM_GROUPS 1 ST:TOTAL_SUBJECTS 6 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:SUBJECT_COMMENTS Plasma #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - PP_A_09_7 Matrix:Plasma .raw file name (POS)=PP_A_09_7.raw .raw file name (NEG)=PP_A_09_7_NEG.raw Emory ID=PlasmaRef_20160726 SUBJECT_SAMPLE_FACTORS - PP_A_09_8 Matrix:Plasma .raw file name (POS)=PP_A_09_8.raw .raw file name (NEG)=PP_A_09_8_NEG.raw Emory ID=PlasmaRef_20160726 SUBJECT_SAMPLE_FACTORS - PP_A_09_9 Matrix:Plasma .raw file name (POS)=PP_A_09_9.raw .raw file name (NEG)=PP_A_09_9_NEG.raw Emory ID=PlasmaRef_20160726 SUBJECT_SAMPLE_FACTORS - PP_A_09_10 Matrix:Plasma .raw file name (POS)=PP_A_09_10.raw .raw file name (NEG)=PP_A_09_10_NEG.raw Emory ID=PlasmaRef_20160726 SUBJECT_SAMPLE_FACTORS - PP_A_09_11 Matrix:Plasma .raw file name (POS)=PP_A_09_11.raw .raw file name (NEG)=PP_A_09_11_NEG.raw Emory ID=PlasmaRef_20160726 SUBJECT_SAMPLE_FACTORS - PP_A_09_12 Matrix:Plasma .raw file name (POS)=PP_A_09_12.raw .raw file name (NEG)=PP_A_09_12_NEG.raw Emory ID=PlasmaRef_20160726 #COLLECTION CO:COLLECTION_SUMMARY N/A CO:SAMPLE_TYPE Plasma CO:STORAGE_CONDITIONS -80˚C #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Plasma: Parent and Sub-Aliquots The CHEAR PlasmaRef_20160726 (500 mL) was SP:SAMPLEPREP_SUMMARY removed from -80°C storage and thawed by storing it at 4°C overnight. The SP:SAMPLEPREP_SUMMARY following day, the thawed plasma was inspected to ensure there was no ice SP:SAMPLEPREP_SUMMARY remaining and the bulk plasma was then mixed in the 4°C room by repeatedly SP:SAMPLEPREP_SUMMARY inverting the container. Parent Aliquots were quickly prepared in the 4°C room SP:SAMPLEPREP_SUMMARY using the Drummond pipet aid and serological pipet. 40 mL plasma aliquots were SP:SAMPLEPREP_SUMMARY transferred to 50 mL tubes in 4°C room and capped immediately and placed on SP:SAMPLEPREP_SUMMARY ice. If needed, the bulk plasma was mixed in between aliquots. The parent SP:SAMPLEPREP_SUMMARY aliquots were labeled appropriately. Sub-Aliquots were prepared on ice at the SP:SAMPLEPREP_SUMMARY bench. The parent aliquots were mixed by inverting the tube thoroughly before SP:SAMPLEPREP_SUMMARY and in between aliquots as needed. 1 mL plasma aliquots were transferred to SP:SAMPLEPREP_SUMMARY cryovials and capped immediately and stored on ice until sample splitting was SP:SAMPLEPREP_SUMMARY competed. The sub aliquots were labeled appropriately. Sub-aliquots were stored SP:SAMPLEPREP_SUMMARY at -80°C. Plasma Aliquots for LCMS platforms: The sub-aliquot of PP_A_09 was SP:SAMPLEPREP_SUMMARY used to prepare aliquots for various LCMS platforms. PP_A_09 was thawed on ice SP:SAMPLEPREP_SUMMARY for 30 – 60 min and vortexed briefly on a vortexer, followed by centrifugation SP:SAMPLEPREP_SUMMARY at 4 °C for 2 minutes at 16,000 rcf. Six aliquots of 60 µL volumes were SP:SAMPLEPREP_SUMMARY prepared for Reverse Phase analysis from PP_A_09. Aliquots were stored at -80 SP:SAMPLEPREP_SUMMARY °C until analysis. Sample preparation for Reverse Phase: The 6 plasma aliquots SP:SAMPLEPREP_SUMMARY were thawed on ice for 30–60 min, and then 50 µL of each sample was SP:SAMPLEPREP_SUMMARY transferred to a new pre-labeled Lo-Bind tube. A volume of 400 µL methanol SP:SAMPLEPREP_SUMMARY containing 0.025 mg/mL tryptophan-d5 as the internal standard was added to SP:SAMPLEPREP_SUMMARY extract metabolites by vortex for 2 mins at 5000 rpm. Sample solution was SP:SAMPLEPREP_SUMMARY centrifuged at 16,000 rcf for 4 min at room temperature to precipitate proteins. SP:SAMPLEPREP_SUMMARY A volume of 350 µL of supernatant was transferred to pre-labeled 2.0 mL Lo-bind SP:SAMPLEPREP_SUMMARY tubes and dried down using a SpeedVac system (LabConco CentriVap) at room SP:SAMPLEPREP_SUMMARY temperature. A volume of 100 µL of Water/Methanol solution (95:5) was added to SP:SAMPLEPREP_SUMMARY reconstitute each sample. Samples were then thoroughly mixed by vortex for 10 SP:SAMPLEPREP_SUMMARY mins at 5000 rpm and then centrifuged at 16,000 rcf for 4 min at room SP:SAMPLEPREP_SUMMARY temperature. The supernatants were transferred to pre-labeled autosampler vials SP:SAMPLEPREP_SUMMARY before analysis. LC-MS analysis: The prepared plasma samples were analyzed by an SP:SAMPLEPREP_SUMMARY LC-MS system comprised of a Waters Acquity UPLC and a Synapt G2-Si ESI-Q-TOF SP:SAMPLEPREP_SUMMARY mass spectrometer. Chromatographic separation was accomplished on an Acquity SP:SAMPLEPREP_SUMMARY HSST3 C18 column (2.1 X 100 mm, 1.8 µm) at 50 °C using a gradient elution SP:SAMPLEPREP_SUMMARY comprised of mobile phase A: 0.1% formic acid-water (v/v), and mobile phase B: SP:SAMPLEPREP_SUMMARY 0.1% formic acid-methanol (v/v), using a flow rate of 0.400 mL/min. The SP:SAMPLEPREP_SUMMARY injection volume was 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 ST000609_AN000933_Results.txt UNITS:Normalized Abundance #END