#METABOLOMICS WORKBENCH czulmer_20150915_9899711_mwtab.txt DATATRACK_ID:378 STUDY_ID:ST000246 ANALYSIS_ID:AN000384 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 Lipid Extraction Efficiency Comparison ST:STUDY_TYPE Lipid Extraction Efficiency Comparison ST:STUDY_SUMMARY Aliquots of Jurkat T-lymphocyte cells were extracted using the Folch or Matyash ST:STUDY_SUMMARY at different total volumes and run in triplicate to calculate the extraction ST:STUDY_SUMMARY for each method. 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 R3-226 Academic Research Building, Department of Biochemistry and Molecular ST:ADDRESS PO Box 100245, Gainesville, FL 32610-0245 ST:EMAIL czulmer@chem.ufl.edu ST:PHONE (352) 392-0515 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 12 #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 SU:SPECIES_GROUP Human #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - folch_high Extraction Method:Folch SUBJECT_SAMPLE_FACTORS - folch_low Extraction Method:Folch SUBJECT_SAMPLE_FACTORS - mtbe_high Extraction Method:Matyash SUBJECT_SAMPLE_FACTORS - mtbe_low Extraction Method:Matyash #COLLECTION CO:COLLECTION_SUMMARY - CO:SAMPLE_TYPE Cells - Jurkat, Clone E6-1 CO:COLLECTION_METHOD Centrifugation CO:COLLECTION_FREQUENCY 1200 rpm CO:COLLECTION_DURATION 5 min CO:VOLUMEORAMOUNT_COLLECTED 10 mL of cell suspension 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 TR:CELL_HARVESTING centrifugation #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were extracted using either the Folch or Matyash method. The total volume SP:SAMPLEPREP_SUMMARY MeOH in each extraction method was varied (0.5 mL or 2.0 mL) while the ratio of SP:SAMPLEPREP_SUMMARY to the other solvents was maintained. SP:SAMPLEPREP_PROTOCOL_FILENAME SOP_LipidExtractionProtocol.pdf SP:PROCESSING_METHOD cell lysis SP:PROCESSING_STORAGE_CONDITIONS on ice SP:EXTRACTION_METHOD Folch or Matyash lipid extraction method SP:SAMPLE_RESUSPENSION 100 μL of IPA SP:SAMPLE_SPIKING internal standards: lysophosphatidylcholine (LPC 17:0 and LPC 19:0), SP:SAMPLE_SPIKING (PC 17:0/17:0 and PC 19:0/19:0), phosphatidylethanolamine (PE 15:0/15:0 and PE SP:SAMPLE_SPIKING phosphatidylserine (PS 14:0/14:0 and PS 17:0/17:0), and phosphatidylglycerol SP:SAMPLE_SPIKING 14:0/14:0 and PG 17:0/17:0), triacylglyceride (TG 15:0/15:0/15:0 and TG 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 Supelco Analytical Titan C18 column CH:METHODS_FILENAME m_lipid_pos_switch_100 CH:COLUMN_PRESSURE 1241 bar (max) CH:COLUMN_TEMPERATURE 30 C CH:FLOW_GRADIENT 32% B at 0 min, 40% B at 1 min, a hold at 40% B until 1.5 min, 45% B at 4 min, CH:FLOW_GRADIENT B at 5 min, 60% B at 8 min, 70% B at 11 min, and 80% B at 14 min CH:FLOW_RATE 0.5 mL/min CH:INJECTION_TEMPERATURE 4 C CH:SAMPLE_INJECTION 2 μL CH:SOLVENT_A 60:40 acetonitrile:water with 0.1% formic acid and 10 mM ammonium formate CH:SOLVENT_B 90:8:2 isopropanol:acetonitrile:water with 0.1% formic acid and 10 mM ammonium CH:ANALYTICAL_TIME 21 min CH:RANDOMIZATION_ORDER 3,10,12,11,8,6,5,4,7,9,2,1 #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Biomedical Mass Spectrometry Lab AN:ACQUISITION_DATE 7/11-12/2014 AN:ACQUISITION_PARAMETERS_FILE QE1_CZU_08_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 30 MS:MASS_ACCURACY <2 ppm MS:SPRAY_VOLTAGE 3.5 kV MS:DESOLVATION_GAS_FLOW .raw MS:SCAN_RANGE_MOVERZ 100-1500 MS:SKIMMER_VOLTAGE 15 V #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples folch_high folch_low mtbe_high mtbe_low Factors Extraction Method:Folch Extraction Method:Folch Extraction Method:Matyash Extraction Method:Matyash PC(17:0/0:0)+H 2367239080.0000 1173754270.0000 1349160679.0000 787697999.8000 PC(17:0/17:0)+H 2347308996.0000 853638932.8000 619440512.5000 271569348.2000 PC(19:0/0:0)+H 2714419478.0000 1279452311.0000 826034899.3000 458158853.3000 PC(19:0/19:0)+H 3341173680.0000 1719493942.0000 1523319934.0000 639825587.2000 PE(15:0/15:0)+H 1251236443.0000 847868482.0000 614580873.4000 301764514.4000 PE(17:0/17:0)+H 248393150.0000 197939185.6000 188878695.4000 74619507.9800 PG(14:0/14:0)+NH4 531616976.5000 258579094.0000 171127089.2000 74833623.0300 PG(17:0/17:0)+NH4 342305741.2000 120563696.2000 110239109.1000 53441496.3700 PS(14:0/14:0)+H 153015056.3000 78399531.4900 28328397.3300 27476440.3400 PS(17:0/17:0)+H 88931263.7900 27861723.9800 12980051.4200 12649316.6200 TG(15:0/15:0/15:0)+NH4 2722439373.0000 2007178451.0000 653556766.3000 385216121.0000 TG(17:0/17:0/17:0)+NH4 2420850736.0000 1928633721.0000 829918104.5000 301486809.6000 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 PC(17:0/0:0)+H 510.3553 UFLORIDA_FEATURE PC(17:0/17:0)+H 762.6007 UFLORIDA_FEATURE PC(19:0/0:0)+H 538.3866 UFLORIDA_FEATURE PC(19:0/19:0)+H 818.6633 UFLORIDA_FEATURE PE(15:0/15:0)+H 720.5537 UFLORIDA_FEATURE PE(17:0/17:0)+H 664.4911 UFLORIDA_FEATURE PG(14:0/14:0)+NH4 768.5749 UFLORIDA_FEATURE PG(17:0/17:0)+NH4 684.481 UFLORIDA_FEATURE PS(14:0/14:0)+H 680.4496 UFLORIDA_FEATURE PS(17:0/17:0)+H 764.5435 UFLORIDA_FEATURE TG(15:0/15:0/15:0)+NH4 866.8171 UFLORIDA_FEATURE TG(17:0/17:0/17:0)+NH4 782.7232 UFLORIDA_FEATURE METABOLITES_END #END