#METABOLOMICS WORKBENCH marneydoran_20171026_114704 DATATRACK_ID:1256 STUDY_ID:ST000887 ANALYSIS_ID:AN001821 PROJECT_ID:PR000616 VERSION 1 CREATED_ON January 10, 2019, 9:06 am #PROJECT PR:PROJECT_TITLE Evaluation of Quenching and Extraction Procedures for Performing Metabolomics in PR:PROJECT_TITLE Acidithiobacillus ferrooxidans PR:PROJECT_TYPE Metabolomic Analysis of At.ferrooxidans at Two Different Growth Phases PR:PROJECT_SUMMARY To perform a Metabolomic Analysis of At. ferrooxidans during its early PR:PROJECT_SUMMARY exponential growth phase as well as its late exponential growth phase. PR:INSTITUTE Laurentian University PR:DEPARTMENT Biochemistry/Chemistry PR:LABORATORY Merritt Lab PR:LAST_NAME Merritt PR:FIRST_NAME Thomas PR:ADDRESS Laurentian University, Sudbury, Ontario, Canada P3E 2C6 PR:EMAIL tmerritt@laurentian.ca PR:PHONE (705)675-1151 ext 2189 PR:FUNDING_SOURCE Natural Science and Engineering Research Council(NSERC), Canada Research PR:FUNDING_SOURCE Chair(CRC) #STUDY ST:STUDY_TITLE Evaluation of Quenching and Extraction Procedures for Performing Metabolomics in ST:STUDY_TITLE Acidithiobacillus ferrooxidans ST:STUDY_SUMMARY To perform a Metabolomic Analysis of At. ferrooxidans during its early ST:STUDY_SUMMARY exponential growth phase as well as its late exponential growth phase. ST:INSTITUTE Laurentian University ST:LAST_NAME Doran ST:FIRST_NAME Marney ST:ADDRESS Laurentian University ST:EMAIL mdoran@laurentian.ca ST:PHONE 8196740310 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Acidithiobacillus ferrooxidans ATCC 23270 SU:TAXONOMY_ID 243159 SU:GENOTYPE_STRAIN Wild Type SU:CELL_BIOSOURCE_OR_SUPPLIER American Type Culture Collection SU:CELL_STRAIN_DETAILS ATCC 23270 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1-Early Exponential Phase Optic Density(OD):0.65 SUBJECT_SAMPLE_FACTORS - 2-Early Exponential Phase Optic Density(OD):0.65 SUBJECT_SAMPLE_FACTORS - 3-Early Exponential Phase Optic Density(OD):0.65 SUBJECT_SAMPLE_FACTORS - 4-Early Exponential Phase Optic Density(OD):0.65 SUBJECT_SAMPLE_FACTORS - 5-Early Exponential Phase Optic Density(OD):0.65 SUBJECT_SAMPLE_FACTORS - 6-Early Exponential Phase Optic Density(OD):0.65 SUBJECT_SAMPLE_FACTORS - 7-Late Exponential Phase Optic Density(OD):0.85 SUBJECT_SAMPLE_FACTORS - 8-Late Exponential Phase Optic Density(OD):0.85 SUBJECT_SAMPLE_FACTORS - 9-Late Exponential Phase Optic Density(OD):0.85 SUBJECT_SAMPLE_FACTORS - 10-Late Exponential Phase Optic Density(OD):0.85 SUBJECT_SAMPLE_FACTORS - 11-Late Exponential Phase Optic Density(OD):0.85 SUBJECT_SAMPLE_FACTORS - 12-Late Exponential Phase Optic Density(OD):0.85 #COLLECTION CO:COLLECTION_SUMMARY Replicate cultures of Acidithiobacillus ferrooxidans (American Type Culture CO:COLLECTION_SUMMARY Collection strain 23270) were grown in 1 L of Tuovinen and Kelly (1973) liquid CO:COLLECTION_SUMMARY medium ([FeSO4·7H2O] = 33.4 g/L; 0.3 % (w/v) (NH4)2SO4; MgSO4·7H2O; K2HPO4, pH CO:COLLECTION_SUMMARY 2.5). Cells were incubated on a gyratory shaker at 180 rpm and 30 °C. Cells CO:COLLECTION_SUMMARY harvested for the metabolomic comparison of two points in the growth curve were CO:COLLECTION_SUMMARY in late exponential growth phase (OD425 = 0.85) or in early exponential growth CO:COLLECTION_SUMMARY phase (OD425 = 0.65). The cells were harvested via centrifugation at 14,000 x g CO:COLLECTION_SUMMARY for 5 min at 4 °C then transferred into a 1.5 mL centrifuge tube. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY After the cells were concentrated into a 1.5ml centrifuge tube, the cellular TR:TREATMENT_SUMMARY metabolism was quenched using 40ul of 60% MeOH and 40% H2O (v/v) with 0.85% TR:TREATMENT_SUMMARY (w/v) of Ammonium Formate, adjusted to pH 2.5 and cooled to -20 °C. The TR:TREATMENT_SUMMARY quenched cells were centrifuged for 5 minutes at 2,319 x g at 4 °C. The cells TR:TREATMENT_SUMMARY were then placed on dry ice, the cell supernatant was removed and an TR:TREATMENT_SUMMARY isopropanol:methanol:water (IMW) (3:3:2) extraction solution that was cooled to TR:TREATMENT_SUMMARY -20 °C was added. These solutions were then vigorously vortexed and then TR:TREATMENT_SUMMARY centrifuged at 15,682 x g for 10 min. The extraction liquid was then collected TR:TREATMENT_SUMMARY and placed at -80 °C until further analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Immediately prior to analysis the extraction solution was evaporated to dryness SP:SAMPLEPREP_SUMMARY and reconstituted in 80:20 Acetonitrile:Water. The samples were normalized based SP:SAMPLEPREP_SUMMARY on Gcdw. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC MS:INSTRUMENT_NAME Bruker MicrOTOF II CH:COLUMN_NAME SeQuant ZIC- pHILIC (150 x 2.1mm, 5um) CH:COLUMN_NAME ZIC-pHILIC 20 x 2.1 mm guard column CH:FLOW_GRADIENT (t= 0-1 minute) 3% A, 97% B; (t= 24 minute) 40% A, 60% B; (t= 28 minute) 40% A, CH:FLOW_GRADIENT 60% B (t= 33 minutes) 3% A, 97% B; (t= 34minutes) 3% A, 97% B. HPLC flow rate CH:FLOW_GRADIENT 0.4 mL/min. CH:FLOW_RATE 0.4ml/min CH:SOLVENT_A 20 mmol ammonium formate solution in LC-MS grade water CH:SOLVENT_B 100% ACN with 0.1% formic acid #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker MicrOTOF II MS:INSTRUMENT_NAME spectrometer MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS None MS:MS_RESULTS_FILE ST000887_AN001821_Results.txt UNITS:m/z-retention time features #END