#METABOLOMICS WORKBENCH czulmer_20150302_9134631_mwtab.txt DATATRACK_ID:212 STUDY_ID:ST000140 ANALYSIS_ID:AN000222 PROJECT_ID:PR000125 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE Mammalian Suspension-Cultured Cellular Metabolomics Workflow PR:PROJECT_TYPE Metabolomics, Lipidomics, Untargeted PR:PROJECT_SUMMARY A workflow was optimized for the sample preparation of a single PR:PROJECT_SUMMARY cell pellet for both metabolomics and lipidomics analysis. Jurkat T-lymphocyte PR:PROJECT_SUMMARY were washed with various rinsing solutions and the lipids extracted using PR:PROJECT_SUMMARY lipid extraction protocols to allow for the most reproducible and quantitative PR:INSTITUTE University of Florida PR:DEPARTMENT Dept. of Chemistry/SECIM PR:LABORATORY Biomedical Mass Spectrometry Lab PR:LAST_NAME Yost PR:FIRST_NAME Richard PR:ADDRESS P.O. Box 117200, Gainesville, FL 32611 PR:EMAIL ryost@aa.ufl.edu PR:PHONE (352) 392-0515 PR:FUNDING_SOURCE JDRF Research Grant #STUDY ST:STUDY_TITLE Cell Rinsing Solution Comparison ST:STUDY_TYPE Cell Rinsing Solution Comparison ST:STUDY_SUMMARY Aliquots of Jurkat T-lymphocyte cells were washed with 5 different rinsing ST:STUDY_SUMMARY (0.3% amm. formate, 0.3% amm. acetate, 0.9% NaCl, 1 M PBS, 100 mM PBS) and ran ST:STUDY_SUMMARY triplicate to monitor the effect of ion suppression on the electrospray ST:STUDY_SUMMARY signal. ST:INSTITUTE University of Florida ST:DEPARTMENT Dept. of Chemistry/SECIM ST:LABORATORY Biomedical Mass Spectrometry Lab ST:LAST_NAME Ulmer ST:FIRST_NAME Candice ST:ADDRESS P.O. Box 117200, Gainesville, FL 32611 ST:EMAIL czulmer@chem.ufl.edu ST:PHONE (352) 392-0515 ST:NUM_GROUPS 5 ST:TOTAL_SUBJECTS 15 #SUBJECT SU:SUBJECT_TYPE Human Cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC - Supplier SU:SUBJECT_COMMENTS P?+10 SU:CELL_PRIMARY_IMMORTALIZED immortalized SU:CELL_PASSAGE_NUMBER P?+10 SU:CELL_COUNTS P?+10 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - amm_acetate rinsing_solution:0.3% ammonium acetate subject_type=Human; cell_line=Jurkat T-lymphocyte SUBJECT_SAMPLE_FACTORS - amm_form rinsing_solution:0.3% ammonium formate subject_type=Human; cell_line=Jurkat T-lymphocyte SUBJECT_SAMPLE_FACTORS - nacl rinsing_solution:0.9% NaCl subject_type=Human; cell_line=Jurkat T-lymphocyte SUBJECT_SAMPLE_FACTORS - hepes rinsing_solution:100 mM HEPES subject_type=Human; cell_line=Jurkat T-lymphocyte SUBJECT_SAMPLE_FACTORS - pbs rinsing_solution:1 M PBS subject_type=Human; cell_line=Jurkat T-lymphocyte #COLLECTION CO:COLLECTION_SUMMARY - CO:SAMPLE_TYPE Jurkat cells, Clone E6-1 CO:COLLECTION_METHOD Centrifugation CO:COLLECTION_FREQUENCY 1200 rpm CO:COLLECTION_DURATION 5 min. CO:STORAGE_CONDITIONS -80°C CO:TISSUE_CELL_QUANTITY_TAKEN 1e6 cells #TREATMENT TR:TREATMENT_SUMMARY - TR:CELL_STORAGE Liquid Nitrogen, 5% DMSO TR:CELL_GROWTH_CONTAINER 10 cm2 culture dish TR:CELL_MEDIA RPMI 1640 TR:CELL_ENVIR_COND 95% humidity, 5% CO2 #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 80% MeOH metabolite extraction SP:SAMPLEPREP_PROTOCOL_ID SOP_PP_02 SP:SAMPLEPREP_PROTOCOL_FILENAME SOP_PP_02_CZU.pdf SP:PROCESSING_METHOD cell lysis SP:PROCESSING_STORAGE_CONDITIONS on ice SP:EXTRACTION_METHOD 80% MeOH SP:SAMPLE_RESUSPENSION 30 ?L 0.1% formic acid in water SP:SAMPLE_SPIKING internal standards: (1) L-tryptophan-2,3,3-d3; (2) creatine-d3; (3) SP:SAMPLE_SPIKING (4) caffeine-d3 SP:CELL_TYPE Jurkat, Clone E6-1 #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Reversed phase CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME ACE Excel 2 C18-PFP (100 x 2.1mm, 2um) CH:METHODS_FILENAME PFP-metabolomics-pos-350 CH:COLUMN_PRESSURE 800 bar (max) CH:COLUMN_TEMPERATURE 30 C CH:FLOW_RATE 0.35 mL/min CH:INJECTION_TEMPERATURE 4 C CH:SAMPLE_INJECTION 5 ?L CH:SOLVENT_A 0.1% formic acid in water CH:SOLVENT_B acetonitrile CH:ANALYTICAL_TIME 18 min CH:RANDOMIZATION_ORDER 3,5,7,2,12,10,15,9,13,14,4,8,6,1,11 #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Biomedical Mass Spectrometry Lab AN:ACQUISITION_DATE 12/7-8/2014 AN:ACQUISITION_PARAMETERS_FILE QE1_CZU_17_SEQUENCE AN:SOFTWARE_VERSION Xcalibur 2.2 AN:OPERATOR_NAME Candice Ulmer AN:DETECTOR_TYPE orbitrap AN:DATA_FORMAT .raw #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:CAPILLARY_TEMPERATURE 320 MS:DRY_GAS_FLOW 45 MS:MASS_ACCURACY <2 ppm MS:SPRAY_VOLTAGE 3.5 kV MS:DATAFORMAT .raw MS:SCAN_RANGE_MOVERZ 70-700 MS:SKIMMER_VOLTAGE 15 V #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples amm_acetate amm_form nacl hepes pbs Factors rinsing_solution:0.3% ammonium acetate rinsing_solution:0.3% ammonium formate rinsing_solution:0.9% NaCl rinsing_solution:100 mM HEPES rinsing_solution:1 M PBS arginine 82331783.8000 31128891.8800 97148010.2600 22627002.0200 20218282.3900 carnitine 14772456.7000 12691064.7000 32000523.6600 2585.1800 9175332.2330 glutamine 1472163.7250 333339.8877 333817.2587 0.0000 121125.9862 guanine 24811119.8600 12239277.2200 13725598.6900 0.0000 9437603.1050 lysine 12310097.2100 4773283.9540 2616477.5780 7002565.3670 1257819.2300 methionine 15530161.7700 24830289.6800 2643381.9230 13997592.8400 179959.8205 nicotinamide 21216139.1200 7186481.7880 19862303.7500 20869738.8900 6389141.3640 phenylalanine 50519131.1000 60219019.4800 433334030.4000 157476421.8000 54695090.6300 proline 86469443.0700 54504149.6000 151641969.9000 3302296.6950 43720506.3300 thiamin 6181644.5680 6809092.6300 19012120.1000 8987762.8830 3621895.8810 tryptophan 10703570.3200 11281477.3600 37715472.4200 20951470.4500 7576416.9890 tyrosine 32197745.4200 31245442.6800 113835764.2000 41472505.7600 22825400.4600 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name moverz_quant ri ri_type pubchem_id inchi_key kegg_id other_id other_id_type arginine 175.119 UFLORIDA_FEATURE carnitine 162.1125 UFLORIDA_FEATURE glutamine 148.0604 UFLORIDA_FEATURE guanine 174.0386 UFLORIDA_FEATURE lysine 147.1128 UFLORIDA_FEATURE methionine 150.0583 UFLORIDA_FEATURE nicotinamide 123.0553 UFLORIDA_FEATURE phenylalanine 166.0863 UFLORIDA_FEATURE proline 116.0706 UFLORIDA_FEATURE thiamin 265.1118 UFLORIDA_FEATURE tryptophan 205.0972 UFLORIDA_FEATURE tyrosine 204.0631 UFLORIDA_FEATURE METABOLITES_END #END