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MB Sample ID: SA188314
| Local Sample ID: | CaOV3_Parent_1 |
| Subject ID: | SU002091 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002091 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CaOV3_Parent_1 | SA188314 | FL023183 | Sensitive | Chemoresistance Status |
Collection:
| Collection ID: | CO002084 |
| Collection Summary: | Two ovarian cancer cell lines were used to generate carboplatin resistant pairs. These were then grown in parallel before untargeted MS metabolomics analysis was applied in the pursuit of characterising metabolome mediated resistance mechanism |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR002103 |
| Treatment Summary: | The human OC cell line: CaOV3, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the OVCAR-5 cell line obtained from Dr. Thomas Hamilton (Fox Chase Cancer Centre, Philadelphia, PA). Both cell lines were authenticated by short tandem repeat (STR) DNA profile in 2020. OVCAR-5 cells were grown in RPMI 1640 media (Sigma Aldrich, St. Louis, MO, USA). Recent reports indi-cate that OVAR-5 might originate from metastatic gastrointestinal cancer and were potentially wrong fully labelled as Ovarian cancer [25]. CaOV3 cells were grown in DMEM media (Sigma Aldrich, St. Louis, MO, USA). Both were cultured with the addi-tion of 10% foetal bovine serum (Bovogen Biologicals, East Keilor, VIC, AUS) supple-mented with 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA) and 1% L-glutamine (Sigma Aldrich, St. Louis, MO, USA). OVCAR-5 and CaOV3 cells were made resistant to CBP after treatment with 6-8 cycles of CBP (50μM, Hospira Austral-ia, Pty Ltd) [26,27]. 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: | Carboplatin |
Sample Preparation:
| Sampleprep ID: | SP002097 |
| Sampleprep 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 ap-proximately 2 mL of liquid nitrogen directly 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 addi-tional 1mL of 100% ice cold methanol was added before 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 repeated 5 times to ensure full extrac-tion 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 tempera-ture, with vacuum of 40mbar and rotor speed of 1000min-1. Before data acquisition samples were resuspended in volumes of 20mM ammonium carbonate and acetonitrile to achieve identical concentration of biological material based on cell number estimate. |
Combined analysis:
| Analysis ID | AN003276 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 RS |
| Column | SeQuant ZIC-HILIC (150 x 4.6mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE |
| Units | Intensity |
Chromatography:
| Chromatography ID: | CH002418 |
| Chromatography Summary: | LCMS data was acquired on Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) coupled with high-performance liquid chromatography (HPLC) system Dionex Ulti-mate® 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 pro-gram 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 25 ºC. The total run time was 32 min with an injection sample vol-ume of 10 µL. Mass spectrometer operated in full scan mode with positive and nega-tive 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. |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | SeQuant ZIC-HILIC (150 x 4.6mm,5um) |
| Column Temperature: | 25 |
| Flow Gradient: | 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. |
| Flow Rate: | 0.3 mL/min |
| Solvent A: | 100% water; 20 mM ammonium carbonate |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003048 |
| Analysis ID: | AN003276 |
| 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 posi-tive mode and 4.0 kV for negative mode, sheath gas was set to 50 and aux gas to 20 ar-bitrary units, capillary temperature 300 °C, probe heater temperature 120 °C. Mixtures of pure authentic standards containing over 320 metabolites were acquired as separate injections and used to confirm retention times. Metabolites confirmed with authentic standards were given the highest confidence MSI level 1. The acquired LCMS data was processed in untargeted fashion using the open-source software IDEOM [30,31]. Default IDEOM parameters were used to elimi-nate unwanted noise and artefact peaks. Putative identification of metabolites was achieved by accurate mass within 3 ppm mass error searching against the Kyoto Ency-clopedia of Genes and Genomes (KEGG), MetaCyc, and LIPIDMAPS databases and others. Despite the washing steps performed in sample preparation it is expected |
| Ion Mode: | POSITIVE |
