{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000403","ANALYSIS_ID":"AN000643","VERSION":"1","CREATED_ON":"May 18, 2016, 3:54 pm"},

"PROJECT":{"PROJECT_TITLE":"Metabolomics-based elucidation of active metabolic pathways in erythrocytes and HSC-derived reticulocytes","PROJECT_SUMMARY":"None","INSTITUTE":"Monash Institute of Pharmaceutical Sciences, Monash University","DEPARTMENT":"Drug Delivery, Disposition and Dynamics","LABORATORY":"Creek lab","LAST_NAME":"Creek","FIRST_NAME":"Darren","ADDRESS":"381 Royal Parade, Parkville, Melbourne, VIC3052, Australia","EMAIL":"Darren.Creek@monash.edu","PHONE":"N/A","FUNDING_SOURCE":"NHMRC"},

"STUDY":{"STUDY_TITLE":"Metabolomics-based elucidation of active metabolic pathways in erythrocytes and HSC-derived reticulocytes","STUDY_TYPE":"Cell type comparison","STUDY_SUMMARY":"Human stem cell derived reticulocytes were compared with mature erythrocytes by metabolomics analysis.","INSTITUTE":"Monash Institute of Pharmaceutical Sciences, Monash University","DEPARTMENT":"Drug Delivery, Disposition and Dynamics","LABORATORY":"Creek lab","LAST_NAME":"Creek","FIRST_NAME":"Darren","ADDRESS":"381 Royal Parade, Parkville, Melbourne, VIC3052, Australia","EMAIL":"Darren.Creek@monash.edu","PHONE":"N/A","NUM_GROUPS":"6","TOTAL_SUBJECTS":"18"},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","CELL_STRAIN_DETAILS":"Reticulocytes","CELL_PRIMARY_IMMORTALIZED":"Primary","CELL_COUNTS":"0.5 x 10e8 per sample"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"CP_RBC37_13C_0",
"Sample ID":"CP_RBC37_13C_0_1",
"Factors":{"Cell type":"Mature erythrocyte","Glucose labelling":"C13 glucose","timepoint":"20 hours"}
},
{
"Subject ID":"CP_RBC37_13C_0",
"Sample ID":"CP_RBC37_13C_0_2",
"Factors":{"Cell type":"Mature erythrocyte","Glucose labelling":"C13 glucose","timepoint":"20 hours"}
},
{
"Subject ID":"CP_RBC37_13C_0",
"Sample ID":"CP_RBC37_13C_0_3",
"Factors":{"Cell type":"Mature erythrocyte","Glucose labelling":"C13 glucose","timepoint":"20 hours"}
},
{
"Subject ID":"CP_RBC37_13C_19",
"Sample ID":"CP_RBC37_13C_19_1",
"Factors":{"Cell type":"Mature erythrocyte","Glucose labelling":"C13 glucose","timepoint":"1 hour"}
},
{
"Subject ID":"CP_RBC37_13C_19",
"Sample ID":"CP_RBC37_13C_19_2",
"Factors":{"Cell type":"Mature erythrocyte","Glucose labelling":"C13 glucose","timepoint":"1 hour"}
},
{
"Subject ID":"CP_RBC37_13C_19",
"Sample ID":"CP_RBC37_13C_19_3",
"Factors":{"Cell type":"Mature erythrocyte","Glucose labelling":"C13 glucose","timepoint":"1 hour"}
},
{
"Subject ID":"CP_RBC37_G_0",
"Sample ID":"CP_RBC37_G_0_1",
"Factors":{"Cell type":"Mature erythrocyte","Glucose labelling":"unlabelled","timepoint":"20 hours"}
},
{
"Subject ID":"CP_RBC37_G_0",
"Sample ID":"CP_RBC37_G_0_2",
"Factors":{"Cell type":"Mature erythrocyte","Glucose labelling":"unlabelled","timepoint":"20 hours"}
},
{
"Subject ID":"CP_RBC37_G_0",
"Sample ID":"CP_RBC37_G_0_3",
"Factors":{"Cell type":"Mature erythrocyte","Glucose labelling":"unlabelled","timepoint":"20 hours"}
},
{
"Subject ID":"CP_Retics37_13C_0",
"Sample ID":"CP_Retics37_13C_0_1",
"Factors":{"Cell type":"Reticulocyte","Glucose labelling":"C13 glucose","timepoint":"20 hours"}
},
{
"Subject ID":"CP_Retics37_13C_0",
"Sample ID":"CP_Retics37_13C_0_2",
"Factors":{"Cell type":"Reticulocyte","Glucose labelling":"C13 glucose","timepoint":"20 hours"}
},
{
"Subject ID":"CP_Retics37_13C_0",
"Sample ID":"CP_Retics37_13C_0_3",
"Factors":{"Cell type":"Reticulocyte","Glucose labelling":"C13 glucose","timepoint":"20 hours"}
},
{
"Subject ID":"CP_Retics37_13C_19",
"Sample ID":"CP_Retics37_13C_19_1",
"Factors":{"Cell type":"Reticulocyte","Glucose labelling":"C13 glucose","timepoint":"1 hour"}
},
{
"Subject ID":"CP_Retics37_13C_19",
"Sample ID":"CP_Retics37_13C_19_2",
"Factors":{"Cell type":"Reticulocyte","Glucose labelling":"C13 glucose","timepoint":"1 hour"}
},
{
"Subject ID":"CP_Retics37_13C_19",
"Sample ID":"CP_Retics37_13C_19_3",
"Factors":{"Cell type":"Reticulocyte","Glucose labelling":"C13 glucose","timepoint":"1 hour"}
},
{
"Subject ID":"CP_Retics37_G_0",
"Sample ID":"CP_Retics37_G_0_1",
"Factors":{"Cell type":"Reticulocyte","Glucose labelling":"unlabelled","timepoint":"20 hours"}
},
{
"Subject ID":"CP_Retics37_G_0",
"Sample ID":"CP_Retics37_G_0_2",
"Factors":{"Cell type":"Reticulocyte","Glucose labelling":"unlabelled","timepoint":"20 hours"}
},
{
"Subject ID":"CP_Retics37_G_0",
"Sample ID":"CP_Retics37_G_0_3",
"Factors":{"Cell type":"Reticulocyte","Glucose labelling":"unlabelled","timepoint":"20 hours"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Peripheral blood mononucleated cells were obtained from blood by Percoll density purification and CD34+ hemopoietic progenitor cells were isolated by magnetic bead separation according to the manufacturer's instructions (Miltenyi Biotec). CD34+ cells were cultured in a three-stage protocol based on the methods of 2d. Initially cells were cultured at 37 °C in a humid atmosphere of 5% CO2 at a density of 1 x 104 cells/mL and then maintained in the range of 2-10 × 105 cells/mL in IMDM (LifeTech) containing 5% (v/v) AB Serum (Interstate Companies Laboratories), 10 μg/mL Insulin (Sigma), 3 U/mL heparin (Pfizer), 200 μg/mL Transferrin (Prospec), 3 U/mL EPO (Eprex). During stage one (days 0-8) this was supplemented with 10 ng/mL SCF (GenScript) and 1 ng/mL IL-3 (R&D systems); during stage two (days 8-11) with 10 ng/mL SCF and additional 800 μg/mL transferrin and stage 3 (days 11-18) with 3 U/mL EPO and additional 800 μg/mL transferrin. Cultured reticulocytes (cRetics) were filtered at day 18 using a PALL WBF leukocyte filter. Isogenic control red blood cells (RBCs) were retained from donor blood, washed in IMDM and stored in saline-adenine-glucose-mannitol solution (SAG-M) at 4°C prior to use. Before analysis, cells were washed and cultured overnight in stage 3-supplemented IMDM (as outlined above).","SAMPLE_TYPE":"Cells","COLLECTION_METHOD":"See summary"},

"TREATMENT":{"TREATMENT_SUMMARY":"None"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Metabolism was quenched by immersion of cultures in an ethanol/dry ice bath to 0-4 °C. Cells were pelleted by centrifugation (10,000 rpm for 1 min) and washed in cold PBS (1 mL). Metabolites were extracted from 1 x 108 RBCs and 0.5 x 108 reticulocytes by addition of 300 µL chloroform/methanol/water (1:3:1 v/v) containing internal standards (CHAPS, CAPS, PIPES and TRIS; 1 µM) and left for 30 mins at 4 °C with periodic mixing and sonication. The number of cells used for RBCs and reticulocytes was based on the mean cell volume of each cell type, and ensured that the total cell pellet volume was equivalent for each sample, allowing direct comparison of metabolite concentrations from these samples. After mixing, cellular debris was removed by centrifugation (16,000 rpm for 10 mins) and the supernatant was kept at -80 °C prior to analysis.","PROCESSING_METHOD":"Lysis with mixing and sonication at 4°C","PROCESSING_STORAGE_CONDITIONS":"on ice or 4°C","EXTRACTION_METHOD":"chloroform/methanol/water (1:3:1 v/v)","EXTRACT_STORAGE":"-80°C","SAMPLE_SPIKING":"internal standards (CHAPS, CAPS, PIPES and TRIS; all at 1 µM) mixed in extraction solvent","CELL_TYPE":"Human stem cell derived reticulocytes and mature erythrocytes"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Untargeted HILIC method","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","COLUMN_NAME":"ZIC-pHILIC (Merck Sequant) column","FLOW_GRADIENT":"linear gradient -time, %B as follows: 0min- 80%, 15min- 50%, 18min- 5%, 21min- 5%, 24min- 80%, 32min- 80%.","FLOW_RATE":"300 μL/min","COLUMN_TEMPERATURE":"25°C","SOLVENT_A":"20 mM ammonium carbonate in water","SOLVENT_B":"100% acetonitrile","INJECTION_TEMPERATURE":"4 C","INTERNAL_STANDARD":"internal standards (CHAPS, CAPS, PIPES and TRIS; all at 1 µM)","SAMPLE_INJECTION":"10 μL"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","OPERATOR_NAME":"Anna Sexton"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","CAPILLARY_TEMPERATURE":"320°C","CAPILLARY_VOLTAGE":"-50V","FRAGMENTATION_METHOD":"None","SPRAY_VOLTAGE":"3.5kV","RESOLUTION_SETTING":"70000","SCAN_RANGE_MOVERZ":"85- 1275 m/z","SKIMMER_VOLTAGE":"-20 V","TUBE_LENS_VOLTAGE":"-70 V"},

"MS_METABOLITE_DATA":{
"Units":"peak height",

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}

}