{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000887","ANALYSIS_ID":"AN001821","VERSION":"1","CREATED_ON":"January 10, 2019, 9:06 am"},

"PROJECT":{"PROJECT_TITLE":"Evaluation of Quenching and Extraction Procedures for Performing Metabolomics in Acidithiobacillus ferrooxidans","PROJECT_TYPE":"Metabolomic Analysis of At.ferrooxidans at Two Different Growth Phases","PROJECT_SUMMARY":"To perform a Metabolomic Analysis of At. ferrooxidans during its early exponential growth phase as well as its late exponential growth phase.","INSTITUTE":"Laurentian University","DEPARTMENT":"Biochemistry/Chemistry","LABORATORY":"Merritt Lab","LAST_NAME":"Merritt","FIRST_NAME":"Thomas","ADDRESS":"Laurentian University, Sudbury, Ontario, Canada P3E 2C6","EMAIL":"tmerritt@laurentian.ca","PHONE":"(705)675-1151 ext 2189","FUNDING_SOURCE":"Natural Science and Engineering Research Council(NSERC), Canada Research Chair(CRC)"},

"STUDY":{"STUDY_TITLE":"Evaluation of Quenching and Extraction Procedures for Performing Metabolomics in Acidithiobacillus ferrooxidans","STUDY_SUMMARY":"To perform a Metabolomic Analysis of At. ferrooxidans during its early exponential growth phase as well as its late exponential growth phase.","INSTITUTE":"Laurentian University","LAST_NAME":"Doran","FIRST_NAME":"Marney","ADDRESS":"Laurentian University","EMAIL":"mdoran@laurentian.ca","PHONE":"8196740310"},

"SUBJECT":{"SUBJECT_TYPE":"Bacteria","SUBJECT_SPECIES":"Acidithiobacillus ferrooxidans ATCC 23270","TAXONOMY_ID":"243159","GENOTYPE_STRAIN":"Wild Type","CELL_BIOSOURCE_OR_SUPPLIER":"American Type Culture Collection","CELL_STRAIN_DETAILS":"ATCC 23270"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"1-Early Exponential Phase",
"Factors":{"Optic Density(OD)":"0.65"}
},
{
"Subject ID":"-",
"Sample ID":"2-Early Exponential Phase",
"Factors":{"Optic Density(OD)":"0.65"}
},
{
"Subject ID":"-",
"Sample ID":"3-Early Exponential Phase",
"Factors":{"Optic Density(OD)":"0.65"}
},
{
"Subject ID":"-",
"Sample ID":"4-Early Exponential Phase",
"Factors":{"Optic Density(OD)":"0.65"}
},
{
"Subject ID":"-",
"Sample ID":"5-Early Exponential Phase",
"Factors":{"Optic Density(OD)":"0.65"}
},
{
"Subject ID":"-",
"Sample ID":"6-Early Exponential Phase",
"Factors":{"Optic Density(OD)":"0.65"}
},
{
"Subject ID":"-",
"Sample ID":"7-Late Exponential Phase",
"Factors":{"Optic Density(OD)":"0.85"}
},
{
"Subject ID":"-",
"Sample ID":"8-Late Exponential Phase",
"Factors":{"Optic Density(OD)":"0.85"}
},
{
"Subject ID":"-",
"Sample ID":"9-Late Exponential Phase",
"Factors":{"Optic Density(OD)":"0.85"}
},
{
"Subject ID":"-",
"Sample ID":"10-Late Exponential Phase",
"Factors":{"Optic Density(OD)":"0.85"}
},
{
"Subject ID":"-",
"Sample ID":"11-Late Exponential Phase",
"Factors":{"Optic Density(OD)":"0.85"}
},
{
"Subject ID":"-",
"Sample ID":"12-Late Exponential Phase",
"Factors":{"Optic Density(OD)":"0.85"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Replicate cultures of Acidithiobacillus ferrooxidans (American Type Culture Collection strain 23270) were grown in 1 L of Tuovinen and Kelly (1973) liquid medium ([FeSO4·7H2O] = 33.4 g/L; 0.3 % (w/v) (NH4)2SO4; MgSO4·7H2O; K2HPO4, pH 2.5). Cells were incubated on a gyratory shaker at 180 rpm and 30 °C. Cells harvested for the metabolomic comparison of two points in the growth curve were in late exponential growth phase (OD425 = 0.85) or in early exponential growth phase (OD425 = 0.65). The cells were harvested via centrifugation at 14,000 x g for 5 min at 4 °C then transferred into a 1.5 mL centrifuge tube.","SAMPLE_TYPE":"Bacterial cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"After the cells were concentrated into a 1.5ml centrifuge tube, the cellular metabolism was quenched using 40ul of 60% MeOH and 40% H2O (v/v) with 0.85% (w/v) of Ammonium Formate, adjusted to pH 2.5 and cooled to -20 °C. The quenched cells were centrifuged for 5 minutes at 2,319 x g at 4 °C. The cells were then placed on dry ice, the cell supernatant was removed and an isopropanol:methanol:water (IMW) (3:3:2) extraction solution that was cooled to -20 °C was added. These solutions were then vigorously vortexed and then centrifuged at 15,682 x g for 10 min. The extraction liquid was then collected and placed at -80 °C until further analysis."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Immediately prior to analysis the extraction solution was evaporated to dryness and reconstituted in 80:20 Acetonitrile:Water. The samples were normalized based on Gcdw."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Bruker MicrOTOF II","COLUMN_NAME":"SeQuant ZIC- pHILIC (150 x 2.1mm, 5um) ZIC-pHILIC 20 x 2.1 mm guard column","FLOW_GRADIENT":"(t= 0-1 minute) 3% A, 97% B; (t= 24 minute) 40% A, 60% B; (t= 28 minute) 40% A, 60% B (t= 33 minutes) 3% A, 97% B; (t= 34minutes) 3% A, 97% B. HPLC flow rate 0.4 mL/min.","FLOW_RATE":"0.4ml/min","SOLVENT_A":"20 mmol ammonium formate solution in LC-MS grade water","SOLVENT_B":"100% ACN with 0.1% formic acid"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Bruker MicrOTOF II spectrometer","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"None","MS_RESULTS_FILE":"ST000887_AN001821_Results.txt UNITS:m/z-retention time features"}

}