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MB Sample ID: SA332076
| Local Sample ID: | A1 |
| Subject ID: | SU003183 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | AGS and AZ-521 |
| Species Group: | AGS and AZ-521 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003183 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | AGS and AZ-521 |
| Species Group: | AGS and AZ-521 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| A1 | SA332076 | FL039284 | Control | Treatment |
Collection:
| Collection ID: | CO003176 |
| Collection Summary: | AGS cells or AZ-521 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum, or minimal essential medium (MEM) containing 10% fetal bovine serum and 5% nonessential amino acids, respectively. AGS cells were seeded at 2x104 cells/well into 96-well plates and incubated overnight. Cultured cells were then incubated with 20 ug/mL purified VacA [activated with by addition of HCl to a pH of 3] in medium supplemented with 5 mM NH4Cl. Following intoxication, the media was removed, and cells were washed with PBS. Cells were detached by incubation with trypsin for 5 minutes and collected via centrifugation at 4°C at 1,000 rpm for 4 minutes. Trypsin was removed, cells were once again washed with PBS, and the cells were then flash-frozen in liquid nitrogen and stored at -70°C. |
| Collection Protocol Filename: | Cell_Culture_Methods.pdf |
| Sample Type: | Cultured cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003192 |
| Treatment Summary: | Cells were incubated in media containing 20 ug/mL of purified VacA toxin and 5 mM ammonium chloride. |
| Treatment Protocol Filename: | Cell_Culture_Methods.pdf |
Sample Preparation:
| Sampleprep ID: | SP003189 |
| Sampleprep Summary: | Briefly, cell pellets were normalized by total protein (200 ug) and the corresponding cell supernatants were normalized by volume (200 uL). Metabolites were extracted with methanol/water 80:20. Heavy labeled phenylalanine-D8 and biotin-D2 were added to individual samples prior to protein precipitation. Following overnight incubation at -80°C, precipitated proteins were pelleted by centrifugation at 10,000 rpm for 10 min and metabolite extracts were transferred into two Eppendorf tubes in equal amounts and dried down in vacuo and stored at -80°C. |
| Sampleprep Protocol Filename: | MS_Methods_Cover_VacA_Mutants.pdf |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN005025 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE |
| Units | time_m/z |
Chromatography:
| Chromatography ID: | CH003797 |
| Methods Filename: | MS_Methods_Cover_VacA_Mutants.pdf |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um) |
| Column Temperature: | 40 |
| Flow Gradient: | 30 min; 95%A, 5%B |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 100% water, 0.1% Formic Acid |
| Solvent B: | 80:20 acetonitrile:water, 0.1% Formic Acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004764 |
| Analysis ID: | AN005025 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometry raw data was imported, processed, normalized, and reviewed using Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK). |
| Ion Mode: | POSITIVE |
