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MB Sample ID: SA184092
| Local Sample ID: | cells-yeast-H3-A110C-01 |
| Subject ID: | SU002032 |
| Subject Type: | Yeast |
| Subject Species: | Saccharomyces cerevisiae |
| Taxonomy ID: | 4932 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002032 |
| Subject Type: | Yeast |
| Subject Species: | Saccharomyces cerevisiae |
| Taxonomy ID: | 4932 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| cells-yeast-H3-A110C-01 | SA184092 | FL022659 | H3A110C | Strain |
Collection:
| Collection ID: | CO002025 |
| Collection Summary: | Please refer to Treatment and Sample Prep sections. |
| Sample Type: | Yeast cells |
Treatment:
| Treatment ID: | TR002044 |
| Treatment Summary: | Yeast strains with wild type or mutant histone H3 were cultured in yeast Synthetic Complete Medium before collection and extraction. As a control and for comparison, a strain with GSH1 deleted is included. GSH1 encodes for gamma glutamylcysteine synthetase, which catalyzes the first step in glutathione biosynthesis. |
Sample Preparation:
| Sampleprep ID: | SP002038 |
| Sampleprep Summary: | To extract metabolites, we washed the cells with ice-cold 150 mM ammonium acetate, pH 7.3, and then added 500 uL 80% methanol and incubated for 20 minutes at -80°C. Cells were then scraped off the plate, vortexed and centrifuged for 10 minutes at maximum speed. We dried 400 uL of the supernatant under vacuum and stored the dried metabolites at -80°C. Dried metabolites were reconstituted in 100 µL of a 50% acetonitrile(ACN) 50% dH20 solution. Samples were vortexed and spun down for 10 min at 17,000g. 70 µL of the supernatant was then transferred to HPLC glass vials. |
Combined analysis:
| Analysis ID | AN003179 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish Horizon |
| Column | SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | Peak Area |
Chromatography:
| Chromatography ID: | CH002351 |
| Chromatography Summary: | Samples were run on a Vanquish (Thermo Scientific) UHPLC system with mobile phase A (20 mM ammonium carbonate, pH 9.7) and mobile phase B (100% Acetonitrile) at a flow rate of 150 µL/min on a SeQuant ZIC-pHILIC Polymeric column (2.1 × 150 mm 5 μm, EMD Millipore) at 35°C. Separation was achieved with a linear gradient from 20% A to 80% A in 20 min followed by a linear gradient from 80% A to 20% A from 20 min to 20.5 min. 20% A was then held from 20.5 min to 28 min. |
| Instrument Name: | Thermo Vanquish Horizon |
| Column Name: | SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
| Column Temperature: | 35°C |
| Flow Gradient: | 100% Acetonitrile |
| Flow Rate: | 150 µL/min |
| Internal Standard: | 10 nM Trifluoromethanesulfonate (extraction buffer) |
| Solvent A: | 100% water; 20 mM ammonium carbonate, pH 9.7 |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002957 |
| Analysis ID: | AN003179 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The UHPLC was coupled to a Q-Exactive (Thermo Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range=(70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard. These “.mzXML” files were imported into the MZmine 2 software package. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well. |
| Ion Mode: | UNSPECIFIED |
