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MB Sample ID: SA351600

Local Sample ID:UM089
Subject ID:SU003329
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

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Subject:

Subject ID:SU003329
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
UM089SA351600FL041039Breast Cancer Cell LineSample source
UM089SA351600FL041039Single_cellSample type

Collection:

Collection ID:CO003322
Collection Summary:Two hours before metabolite extraction, the cell culture medium was replaced with fresh medium. Single cell and cell cluster states were collected and centrifuged at 800 × g for 3 min to remove culture media. Then, the cells were resuspended in 1 mL of precooled PBS (4°C) and transferred into a 1.5 mL eppendorf tube. After removing supernatant by centrifugation at 800 × g for 3 min, the tube containing cell pellet was immediately incubated in liquid nitrogen for 1 min to quench enzyme activity and stop the degradation of metabolites.
Collection Protocol Filename:WZC_Collection_protocol.pdf
Sample Type:Cultured cells

Treatment:

Treatment ID:TR003338
Treatment Summary:MDA-MB-231 cells and MDA-MB-453 were maintained in L-15 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a free gas exchange with atmospheric air.

Sample Preparation:

Sampleprep ID:SP003336
Sampleprep Summary:300 μL of H2O was then added to the tube, and the cells were fully lysed by an ultrasonic cell disruptor for 10 min at 4°C. After mixing, 20 μL of suspension was collected for protein quantification and subsequent data normalization. Then, methanol (precooled to −80°C) was added into the tube to make 80% methanol solution (v/v). The tube was vortexed vigorously for 5 min and centrifuged at 14,000 × g for 10 min at 4°C to remove the cell debris. Afterward, the metabolite-containing supernatant was transferred to a new tube on ice. The supernatant was evaporated to dryness in a vacuum concentrator and reconstituted in 100 μL of methanol: H2O (1:1, v/v), which was centrifuged again at 14000 × g for 15 min at 4°C to remove insoluble debris.
Sampleprep Protocol Filename:Sampleprep_protocol.pdf

Combined analysis:

Analysis ID AN005262 AN005263 AN005264 AN005265
Analysis type MS MS MS MS
Chromatography type HILIC HILIC Reversed phase Reversed phase
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000
Column Agilent InfinityLab Poroshell 120 HILIC-Z (100 x 2.1mm, 2.7um) Agilent InfinityLab Poroshell 120 HILIC-Z (100 x 2.1mm, 2.7um) Phenomenex Kinetex C18 (100 x 2.1mm,2.6um) Phenomenex Kinetex C18 (100 x 2.1mm,2.6um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE NEGATIVE POSITIVE NEGATIVE
Units Peak area Peak area Peak area Peak area

Chromatography:

Chromatography ID:CH003983
Methods Filename:Chromatography_methods.pdf
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Agilent InfinityLab Poroshell 120 HILIC-Z (100 x 2.1mm, 2.7um)
Column Temperature:40
Flow Gradient:85% B (0 min) to 85% B (1 min) to 65% B (12 min) to 40% B (12.1 min) to 40% B (15 min) to 85% B (15.1 min) to 85% B (20 min), the flow gradient increases linearly between the time points mentioned.
Flow Rate:0.3 mL/min
Solvent A:100% Water; 25 mM Ammonium acetate; 25 mM Ammonium hydroxide
Solvent B:100% Acetonitrile
Chromatography Type:HILIC
  
Chromatography ID:CH003984
Methods Filename:Chromatography_methods.pdf
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Phenomenex Kinetex C18 (100 x 2.1mm,2.6um)
Column Temperature:40
Flow Gradient:10% B (0 min) to 30% B (1 min) to 95% B (19 min) to 95% B (20 min) to 10% B (20.1 min) to 10% B (23 min), the flow gradient increases linearly between the time points mentioned.
Flow Rate:0.4 mL/min
Solvent A:100% Water; 0.1% Formic acid
Solvent B:100% Acetonitrile; 0.1% Formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS004993
Analysis ID:AN005262
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All operations and acquisitions were controlled by Xcalibur 2.0.7 (Thermo Fisher Scientific, Bremen, Germany).UPLC-HRMS data were analyzed using MS-DIAL software after conversion to mzML file format using MSConvert (Version 3.0, ProteiWizard).
Ion Mode:POSITIVE
Analysis Protocol File:MS_analysis_protocol.pdf
  
MS ID:MS004994
Analysis ID:AN005263
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All operations and acquisitions were controlled by Xcalibur 2.0.7 (Thermo Fisher Scientific, Bremen, Germany).UPLC-HRMS data were analyzed using MS-DIAL software after conversion to mzML file format using MSConvert (Version 3.0, ProteiWizard).
Ion Mode:NEGATIVE
Analysis Protocol File:MS_analysis_protocol.pdf
  
MS ID:MS004995
Analysis ID:AN005264
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All operations and acquisitions were controlled by Xcalibur 2.0.7 (Thermo Fisher Scientific, Bremen, Germany).UPLC-HRMS data were analyzed using MS-DIAL software after conversion to mzML file format using MSConvert (Version 3.0, ProteiWizard).
Ion Mode:POSITIVE
Analysis Protocol File:MS_analysis_protocol.pdf
  
MS ID:MS004996
Analysis ID:AN005265
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All operations and acquisitions were controlled by Xcalibur 2.0.7 (Thermo Fisher Scientific, Bremen, Germany).UPLC-HRMS data were analyzed using MS-DIAL software after conversion to mzML file format using MSConvert (Version 3.0, ProteiWizard).
Ion Mode:NEGATIVE
Analysis Protocol File:MS_analysis_protocol.pdf
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