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MB Sample ID: SA201531
| Local Sample ID: | CaOV3_CBPR_3 |
| Subject ID: | SU002189 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Genotype Strain: | OVCAR-5 and CaOV3 cell lines |
| Gender: | Not applicable |
| Cell Biosource Or Supplier: | CaOV3 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). OVCAR-5 cell line obtained from Dr. Thomas Hamilton (Fox Chase Cancer Centre, Philadelphia, PA). |
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Subject:
| Subject ID: | SU002189 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Genotype Strain: | OVCAR-5 and CaOV3 cell lines |
| Gender: | Not applicable |
| Cell Biosource Or Supplier: | CaOV3 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). OVCAR-5 cell line obtained from Dr. Thomas Hamilton (Fox Chase Cancer Centre, Philadelphia, PA). |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CaOV3_CBPR_3 | SA201531 | FL024521 | CaOV3 cell line CBPR mutation | conditions |
Collection:
| Collection ID: | CO002182 |
| Collection Summary: | Cells were maintained at 60-80% confluence for 3 passages before being plated in 10cm dishes. Cell numbers were estimated from an additional dish with the same number of cells at seeding. Media was aspirated and cells were washed three times with 3mL warmed PBS. Metabolic arrest was achieved through the addition of approximately 2 mL of liquid nitrogen directly to cells ensuring that the surface of the plate was covered before plates were placed onto dry ice. |
| Sample Type: | Cultured cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002201 |
| Treatment Summary: | OVCAR-5 and CaOV3 cells were made resistant to carboplatinumv (CBP) after treatment with 6-8 cycles of CBP (50μM, Hospira Australia, Pty Ltd). Resistance to CBP was measured regularly and CBPR cell lines were seen to be at least two-fold more resistant to CBP than their parental partners through the following experiments. |
| Treatment Compound: | 50μM carboplatinum, Hospira Australia, Pty Ltd |
| Treatment Route: | in culture media |
| Treatment Dose: | 6-8 cycles of CBP 50μM |
Sample Preparation:
| Sampleprep ID: | SP002195 |
| Sampleprep Summary: | Media was aspirated and cells were washed three times with 3mL warmed PBS. Metabol-ic arrest was achieved through the addition of approximately 2 mL of liquid nitrogen di-rectly to cells ensuring that the surface of the plate was covered before plates were placed onto dry ice. Metabolite extraction was achieved through the addition of 1mL 100% ice cold methanol. Cells were lifted off of the plate using an ice cold cell scraper and transferred to a 2mL Eppendorf. An additional 1mL of 100% ice cold methanol was added be-fore snap freezing by immersion in liquid nitrogen for 3 minutes. This was followed by thawing on dry ice and vortexing to resuspend contents. Freeze/thaw process was repeat-ed 5 times to ensure full extraction of metabolites. Samples were centrifuged at 16000g at -9⁰C for 5 minutes and the supernatant was retained. The pellet was resuspended in 500uL of 100% ice cold methanol and freeze/thaw in liquid nitrogen was repeated 5 times. This sample was centrifuged at 16000g at -9⁰C for 5 minutes and the supernatant was retained and combined with previously retained supernatant. The samples were then dried in a SpeedVac Vacuum Concentrator (John Morris Scientific, RVC 2-18) at room temperature, with vacuum of 40mbar and rotor speed of 1000min-1. Before data acquisition samples were resuspended in appropriate volumes of methanol as per cell number. |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | MeOH |
| Extract Storage: | -80℃ |
| Sample Resuspension: | MeOH |
Combined analysis:
| Analysis ID | AN003439 | AN003440 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
| Column | SeQuant ZIC-pHILIC (150 x 4.2mm,5um) | SeQuant ZIC-pHILIC (150 x 4.2mm,5um) |
| 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: | CH002541 |
| Chromatography Summary: | LCMS data was acquired on Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) coupled with high-performance liquid chromatography (HPLC) system Dionex Ultimate® 3000 RS (Thermo Fisher). Chromatographic separation was performed on a ZIC-pHILIC column (5 µm, polymeric, 150 × 4.6 mm, SeQuant®, Merck). The mobile phase (A) was 20 mM ammonium carbonate and (B) acetonitrile. The gradient program started at 80% B and was reduced to 50% B over 15 min, then reduced from 50% B to 5% B over 3 min, followed by wash with 5% B for another 3 min, and finally 8 min re-equilibration with 80% B. The flow rate was 0.3 mL/min and column compartment temperature was 40ºC. The total run time was 32 min with an injection sample volume of 10 µL. |
| Methods Filename: | Metabolomics_pHILIC_Parkville_v1.meth |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | SeQuant ZIC-pHILIC (150 x 4.2mm,5um) |
| Column Pressure: | 60 bar at starting conditions |
| Column Temperature: | 25 C |
| Flow Gradient: | 0 min - 80%B, 15 min - 50%B, 18 min - 5%B, 21 min - 5%B, 24 min - 80%B, 32 min - 80%B |
| Flow Rate: | 0.3 ml/min |
| Injection Temperature: | 4 C |
| Internal Standard: | CAPS, CHAPS, PIPES |
| Sample Injection: | 10 uL |
| Solvent A: | 100% water; 20 mM ammonium formate |
| Solvent B: | 100% acetonitrile |
| Analytical Time: | 32 min |
| Capillary Voltage: | 3.5 kV |
| Oven Temperature: | 25 C |
| Washing Buffer: | syringe wash 50% IPA |
| Weak Wash Solvent Name: | 10% MeOH |
| Strong Wash Solvent Name: | 10% MeOH |
| Sample Loop Size: | 25 uL |
| Sample Syringe Size: | 25 uL |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003202 |
| Analysis ID: | AN003439 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometer operated in full scan mode with positive and negative polarity switching at 35000 resolution at 200 m/z with detection range of 85 to 1, 275 m/z in full scan mode. Electro-spray ionization source (HESI) was set to 3.5 kV voltage for positive mode and 4.0 kV for negative mode, sheath gas was set to 50 and aux gas to 20 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 300 C |
| Capillary Voltage: | 4 kV |
| Collision Energy: | NA |
| Collision Gas: | NA |
| Ion Source Temperature: | 120 C |
| Mass Accuracy: | 1-2 ppm |
| Automatic Gain Control: | 1e6 |
| Dataformat: | profile |
| Acquisition Parameters File: | Metabolomics_pHILIC_Parkville_v1.pdf |
| Analysis Protocol File: | PQMS3-MBPF-WIN-0501_analysis.pdf |
| MS ID: | MS003203 |
| Analysis ID: | AN003440 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometer operated in full scan mode with positive and negative polarity switching at 35000 resolution at 200 m/z with detection range of 85 to 1, 275 m/z in full scan mode. Electro-spray ionization source (HESI) was set to 3.5 kV voltage for positive mode and 4.0 kV for negative mode, sheath gas was set to 50 and aux gas to 20 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C. |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 300 C |
| Capillary Voltage: | 3.5 kV |
| Collision Energy: | NA |
| Collision Gas: | NA |
| Ion Source Temperature: | 120 C |
| Mass Accuracy: | 1-2 ppm |
| Automatic Gain Control: | 1e6 |
| Dataformat: | profile |
| Acquisition Parameters File: | Metabolomics_pHILIC_Parkville_v1.pdf |
| Analysis Protocol File: | PQMS3-MBPF-WIN-0501_analysis.pdf |
