Summary of study ST000539

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000315. The data can be accessed directly via it's Project DOI: 10.21228/M8002N This work is supported by NIH grant, U2C- DK119886.

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Study IDST000539
Study TitleMetabolomics-based elucidation of active metabolic pathways in erythrocytes and HSC-derived reticulocytes (part II)
Study TypeCell type comparison
Study SummaryHuman stem cell derived reticulocytes were compared with mature erythrocytes by metabolomics analysis.
Institute
Monash University
DepartmentMonash Institute of Pharmaceutical Sciences, Drug Delivery, Disposition and Dynamics
LaboratoryCreek lab
Last NameCreek
First NameDarren
Address381 Royal Parade, Parkville, Melbourne, VIC3052, Australia
EmailDarren.Creek@monash.edu
PhoneN/A
Submit Date2017-01-23
Num Groups6
Total Subjects18
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2017-07-10
Release Version1
Darren Creek Darren Creek
https://dx.doi.org/10.21228/M8002N
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000315
Project DOI:doi: 10.21228/M8002N
Project Title:Metabolomics-based elucidation of active metabolic pathways in erythrocytes and HSC-derived reticulocytes
Project Summary:None
Institute:Monash University
Department:Monash Institute of Pharmaceutical Sciences, 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

Subject:

Subject ID:SU000561
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
Species Group:Human

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Cell type Glucose labelling timepoint
SA027945CP_RBC36_13C_19_3Mature erythrocyte C13 glucose 1 hour
SA027946CP_RBC36_13C_19_2Mature erythrocyte C13 glucose 1 hour
SA027947CP_RBC36_13C_19_1Mature erythrocyte C13 glucose 1 hour
SA027948CP_RBC36_13C_0_1Mature erythrocyte C13 glucose 20 hours
SA027949CP_RBC36_13C_0_2Mature erythrocyte C13 glucose 20 hours
SA027950CP_RBC36_13C_0_3Mature erythrocyte C13 glucose 20 hours
SA027951CP_RBC36_G_0_3Mature erythrocyte unlabelled 20 hours
SA027952CP_RBC36_G_0_1Mature erythrocyte unlabelled 20 hours
SA027953CP_RBC36_G_0_2Mature erythrocyte unlabelled 20 hours
SA027954CP_Retics36_13C_19_1Reticulocyte C13 glucose 1 hour
SA027955CP_Retics36_13C_19_2Reticulocyte C13 glucose 1 hour
SA027956CP_Retics36_13C_19_3Reticulocyte C13 glucose 1 hour
SA027957CP_Retics36_13C_0_1Reticulocyte C13 glucose 20 hours
SA027958CP_Retics36_13C_0_3Reticulocyte C13 glucose 20 hours
SA027959CP_Retics36_13C_0_2Reticulocyte C13 glucose 20 hours
SA027960CP_Retics36_G_0_3Reticulocyte unlabelled 20 hours
SA027961CP_Retics36_G_0_2Reticulocyte unlabelled 20 hours
SA027962CP_Retics36_G_0_1Reticulocyte unlabelled 20 hours
Showing results 1 to 18 of 18

Collection:

Collection ID:CO000555
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 ID:TR000575
Treatment Summary:None

Sample Preparation:

Sampleprep ID:SP000568
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

Combined analysis:

Analysis ID AN000818 AN000819
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS
Column ZIC-pHILIC (Merck Sequant) column ZIC-pHILIC (Merck Sequant) column
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units peak height peak height

Chromatography:

Chromatography ID:CH000585
Chromatography Summary:Untargeted HILIC method
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:ZIC-pHILIC (Merck Sequant) column
Column Temperature:25°C
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
Injection Temperature:4 C
Internal Standard:internal standards (CHAPS, CAPS, PIPES and TRIS; all at 1 µM)
Sample Injection:10 μL
Solvent A:20 mM ammonium carbonate in water
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS000719
Analysis ID:AN000818
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:POSITIVE
Capillary Temperature:300°C
Capillary Voltage:+50V
Fragmentation Method:None
Spray Voltage:4.0kV
Resolution Setting:35000
Scan Range Moverz:85- 1275 m/z
Skimmer Voltage:+20 V
Tube Lens Voltage:+70 V
  
MS ID:MS000720
Analysis ID:AN000819
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:NEGATIVE
Capillary Temperature:300°C
Capillary Voltage:-50V
Fragmentation Method:None
Spray Voltage:3.5kV
Resolution Setting:35000
Scan Range Moverz:85- 1275 m/z
Skimmer Voltage:-20 V
Tube Lens Voltage:-70 V
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