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MB Sample ID: SA164971
| Local Sample ID: | S7R2 |
| Subject ID: | SU001853 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Female |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001853 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| S7R2 | SA164971 | FL019732 | HgMe | Treatment |
Collection:
| Collection ID: | CO001846 |
| Collection Summary: | At the endpoint of the experiment, cells were washed gently with 150 mM ammonium acetate buffer and scraped quickly with a rubber-tipped cell scraper. Subsequently, 500 uL chilled 80% methanol was added and the scraped cells were sonicated using a bioruptor (Diagenode, Philadelphia, PA, USA) in 4 °C water bath during 10 cycles (30s on and 30s off). After that, the sample was placed at -20 °C for 3 h, followed by 10 min of centrifugation at 14000 g at 4 °C. The supernatant containing the metabolite was collected, dried, and stored at -80 °C until LC-HRMS analysis. |
| Sample Type: | HEK cells |
Treatment:
| Treatment ID: | TR001866 |
| Treatment Summary: | About 1 million cells were seeded in a 25 cm2 flask. When cells reached about 50% confluence, the experimental group was changed with medium containing 7.5 uM methylmercury and the control group was changed with fresh culture medium. After 48 h treatment, cells were harvested. |
| Cell Growth Container: | 25 cm2 flask |
| Cell Media: | DMEM |
| Cell Harvesting: | 90% confluence |
Sample Preparation:
| Sampleprep ID: | SP001859 |
| Sampleprep Summary: | cells were washed gently with 150 mM ammonium acetate buffer and scraped quickly with a rubber-tipped cell scraper. Subsequently, 500 uL chilled 80% methanol was added and the scraped cells were sonicated using a bioruptor (Diagenode, Philadelphia, PA, USA) in 4 °C water bath during 10 cycles (30s on and 30s off). After that, the sample was placed at -20 °C for 3 h, followed by 10 min of centrifugation at 14000 g at 4 °C. The supernatant containing the metabolite was dried by speedvac and stored at -80 °C until LC-HRMS analysis.The residual protein pellet was dissolved in protein extraction buffer and quantified for normalization. |
| Processing Storage Conditions: | 4℃ |
| Extract Storage: | -80℃ |
| Sample Resuspension: | Sampe were reconstituted in 50% ACN. The volume was adjusted by the protein concentration. |
Combined analysis:
| Analysis ID | AN002883 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 RS |
| Column | Thermo Accucore HILIC (100 x 2.1mm,2.6um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE |
| Units | arbitray unit |
Chromatography:
| Chromatography ID: | CH002138 |
| Chromatography Summary: | The UHPLC system was equipped with a binary pump, an autosampler and a column thermostat. The autosampler was set at 8 °C. The column oven temperature was 30 °C. Mobile phase A was 98% acetonitrile, 2% water and 0.1% formic acid while mobile phase B was 98% water, 2% acetonitrile, 30 mM ammonium formate and 0.1% formic acid. The mobile phase was freshly prepared before use. The optimized stepwise linear gradient (10% B in the first 2 min, 10% - 30% B in the next 5 min, followed by 30% - 60% in 3 min, and finally 60% - 90% in 5 min. |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Thermo Accucore HILIC (100 x 2.1mm,2.6um) |
| Column Temperature: | 25 |
| Solvent A: | 98% acetonitrile/2% water; 0.1% formic acid |
| Solvent B: | 98% water/2% acetonitrile; 0.1% formic acid; 30 mM ammonium formate |
| Sample Loop Size: | 10 |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002676 |
| Analysis ID: | AN002883 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The MS acquisition parameters were as follows: spray voltage, 3.5 kV; capillary temperature, 320 °C; sheath gas flow rate, 45; auxiliary gas flow rate, 25; heater temperature 350 °C; AGC, 3×106; maximum injection time, 200 ms; mass scan range, 50–750; full MS resolution, 70,000 FWHM at m/z 200; spectrum data type, profile. The raw LC-HRMS files for the same study were processed in one batch using the label-free differential analysis software (SIEVE 2.2, Thermo Scientific), in which the ChromAlign algorithm was used. The key parameters for the feature extraction were as follows: signal to background noise,>3; Mzstep accuracy, 10 ppm, minimum peak intensity 200,000; minimum peak scan points, 5; minimum isotopes,1. |
| Ion Mode: | POSITIVE |
