Return to study ST003134 main page
MB Sample ID: SA339338
| Local Sample ID: | NC-7 |
| Subject ID: | SU003251 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003251 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| NC-7 | SA339338 | FL040195 | Erastin-resis SNU-674 cells | Sample source |
| NC-7 | SA339338 | FL040195 | shRNA-NC | transfected |
Collection:
| Collection ID: | CO003244 |
| Collection Summary: | SNU-668 Erastin-resistant cells were were cultured for 48-72 h in advanced RPMI-1640 medium (Gibco) without supplements. |
| Sample Type: | SNU-668 Erastin-resistant cells |
Treatment:
| Treatment ID: | TR003260 |
| Treatment Summary: | Erastin-resis SNU-668 cells transfected with shRNA-NC or shRNA-SOX13 |
Sample Preparation:
| Sampleprep ID: | SP003258 |
| Sampleprep Summary: | For untargeted metabolomics, a total of 24 samples were analyzed (n=12 Erastinresis SNU-668 cells transfected with shRNA-NC, n=12 Erastinresis SNU-668 cells transfected with shRNA-SOX13). 2 × 105 cells of adherent cells were harvested in six-well plates. When collected, cells were washed by cold PBS buffer twice and immediately quenched in liquid nitrogen. Tumor samples were weighed and pulverized. All samples were lysed in 1 ml of −80°C extraction solvent (80% methanol/water). After centrifugation (20,000g, 4°C, 15 min), supernatant was transferred to a new tube, and samples were dried using a vacuum centrifugal concentrator. Blood samples from patients and mice were collected into BD Vacutainer blood collection tubes and placed on ice. Serum was isolated by centrifugation (15,000g, 4°C, 10 min), and aliquots of 100 μl of supernatant were frozen immediately at −80°C. Metabolites were reconstituted in 150 μl of 80% acetonitrile/water, vortexed, and centrifuged to remove insoluble material. All samples were stored at −80°C before LC-MS/MS analysis. |
Combined analysis:
| Analysis ID | AN005144 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Agilent 1260 |
| Column | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE |
| Units | m/z |
Chromatography:
| Chromatography ID: | CH003894 |
| Chromatography Summary: | Samples were separated on an amide column, using mobile phase A consists of water mixed with 25 mM ammonium acetate and 25 mM Ammonium hydroxide and mobile phase B ACN. The injection volume was 4 µL and flow rate was 0.4 ml/min. 1. The generic HPLC gradient was listed in Table 1: 2. |
| Methods Filename: | FUSCC_methods.pdf |
| Instrument Name: | Agilent 1260 |
| Column Name: | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) |
| Column Temperature: | 350 |
| Flow Gradient: | 0.0 min 10% A; 1.0 min 10% A; 11.0 min 13% A; 14.0 min 20% A; 16.5 min 30% A; 18.5 min 50% A; 20.5 min 80% A; 25.0 min 80% A; 25.1 min 10% A; 34.0 min 10% A |
| Flow Rate: | 0.4 ml/min |
| Solvent A: | 100% water; 25mM ammonium acetate; 25mM ammonium hydroxide |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004880 |
| Analysis ID: | AN005144 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS analysis was carried out on the Q-Exactive MS/MS in both positive and negative ion modes. 1) Set the relevant tuning parameters for the probe as listed: aux gas heater temperature, 400 °C; sheath gas, 40; auxiliary gas, 13; spray voltage, 3.5 kV for positive mode and negative mode. Set the capillary temperature at 350 °C, and S-lens at 55. 2) Build a DDA method as follows: Full scan range: 60 to 900 (m/z); resolution for MS1 and ddMS2: 70,000 and 17,500 respectively; maximum injection time for MS1 and ddMS2: 100 ms and 45 ms; automatic gain control (AGC) for MS1 and ddMS2: 3e6 and 2e5; isolation window: 1.6 m/z; normalized collision energies (NCE): 10, 17, 25 or 30, 40, 50. 3) Build a full scan method as follows: Full scan range: 60 to 900 (m/z); resolution: 140,000; maximum injection time: 100ms; automatic gain control (AGC): 3e6 ions. Raw files were submitted to Thermo Compound Discover 2.1, (CD), and processed with Untargeted Metabolomics workflow with minor modification to find and identify the differences between samples: Performs retention time alignment, unknown compound detection, and compound grouping across all samples. Predicts elemental compositions for all compounds, fills gaps across all sam ples, and hides chemical background (using Blank samples). Identifies compounds using mzCloud (ddMS2) and ChemSpider (formula or exact mass). Also performs similarity search for all com pounds with ddMS2 data using mzCloud. Maps compounds to biological pathways using KEGG database For retention time alignment, the max time shift was 2 mins, and a tolerance of 0.5 min was used for grouping unknown compounds. Mass tolerance were set as 10 ppm for feature detection and 5 ppm for compound annotation. The exact mass of each feature was submitted to ChemSpider with 4 databases selected (BioCyc; Human Metabolome Database; KEGG; LipidMAPS). Results from Compound Discover, the compound table, was exported as .xsls file, and then analysed with R. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | FUSCC_methods.pdf |
