#METABOLOMICS WORKBENCH csclendinen_20140910_9105751_mwtab.txt DATATRACK_ID:149 STUDY_ID:ST000098 ANALYSIS_ID:AN000158 PROJECT_ID:PR000090 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE 13C Metabolomics: NMR and IROA for Unknown Identification PR:PROJECT_SUMMARY Demonstrate a method using high-sensitivity NMR to identify an unknown PR:PROJECT_SUMMARY 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:ADDRESS - PR:EMAIL aedison@ufl.edu PR:PHONE 352-392-4535 #STUDY ST:STUDY_TITLE Heatshock response of C. elegans using IROA (II) ST:STUDY_SUMMARY - ST:INSTITUTE University of Florida ST:DEPARTMENT Department of Biochemistry and Molecular Biology / SECIM 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 352-392-4535 ST:NUM_GROUPS 1 ST:TOTAL_SUBJECTS 1 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Caenorhabditis elegans SU:GENOTYPE_STRAIN N2 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 HSP HSP_F9_ms2_neg.raw Fractionation:fraction 4-4.5 min SUBJECT_SAMPLE_FACTORS HSP HSP_F9_pos.raw Fractionation:fraction 4-4.5 min SUBJECT_SAMPLE_FACTORS HSP HSP_F10_ms2_neg.raw Fractionation:fraction 4.5-5 min SUBJECT_SAMPLE_FACTORS HSP HSP_F10_pos.raw Fractionation:fraction 4.5-5 min SUBJECT_SAMPLE_FACTORS HSP HSP_ms2_pos.raw Fractionation:Unfractionated SUBJECT_SAMPLE_FACTORS HSP HSP_pos.raw Fractionation:Unfractionated #COLLECTION CO:COLLECTION_SUMMARY Samples were already collected. CO:COLLECTION_PROTOCOL_FILENAME NMR_Methods.docx;LCMSMethod.docx #TREATMENT TR:TREATMENT_SUMMARY No further treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were mixed together and resuspended in 120 ?L of water. SP:SAMPLEPREP_PROTOCOL_FILENAME NMR_Methods.docx;LCMSMethod.docx #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were injected onto an Waters 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_RATE 300 L/min CH:INJECTION_TEMPERATURE 4 C 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 Timothy Garrett AN:OPERATOR_NAME Timothy Garrett AN:DETECTOR_TYPE Thermo Scientific Q-Exactive Orbitrap/MS2 Positive #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS HSP_ms2_pos.raw MS:FRAGMENTATION_METHOD Full MS/DD-MS2 (TOPN) MS:RESOLUTION_SETTING 70,000, 17,500 MS:SCAN_RANGE_MOVERZ 70-1000 MS:MS_COMMENTS Samples were analyzed using a mass range of 70-1000 in positive and negative MS:MS_COMMENTS modes, externally calibrated, using a Thermo Scientific Q-Exactive Orbitrap MS:MS_COMMENTS spectrometer equipped with a Dionex UltiMate 3000 RS autosampler and pump. The MS:MS_COMMENTS was equipped with a Heated Electrospray Ionization (HESI) source which operated MS:MS_COMMENTS a spray temperature of 350 ?C, a spray voltage of 3.5 kV, and sheath and MS:MS_COMMENTS gas flow rates of 50 and 10 arbitrary units, respectively. The capillary MS:MS_COMMENTS was held at 325 ?C, and the S-lens RF Level was set to 40%. The FR resolution MS:MS_COMMENTS set to 70,000 at m/z 200. The accuracy achieved was routinely less than 1.5 MS:MS_COMMENTS In addition to full MS, data-dependent MS/MS was performed on the 10 most MS:MS_COMMENTS peaks with an exclusion period of 10 seconds (40 normalized collision energy). MS:MS_COMMENTS was performed using a Dionex UltiMate 3000 fraction collector by triggering MS:MS_COMMENTS 30 seconds. #END