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MB Sample ID: SA212646
Local Sample ID: | MCT_EV2_SD |
Subject ID: | SU002318 |
Subject Type: | Yeast |
Subject Species: | Saccharomyces cerevisiae |
Taxonomy ID: | 4932 |
Genotype Strain: | BY4743 (MATa/\textalpha, his3/his3, leu2/leu2, ura3/ura3, met15/MET15, lys2/LYS2) |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002318 |
Subject Type: | Yeast |
Subject Species: | Saccharomyces cerevisiae |
Taxonomy ID: | 4932 |
Genotype Strain: | BY4743 (MATa/\textalpha, his3/his3, leu2/leu2, ura3/ura3, met15/MET15, lys2/LYS2) |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
MCT_EV2_SD | SA212646 | FL025949 | BY4743 | Strain |
MCT_EV2_SD | SA212646 | FL025949 | mct1 deletion | Genotype |
MCT_EV2_SD | SA212646 | FL025949 | empty vector | Experimental Factor 1 - Vector |
MCT_EV2_SD | SA212646 | FL025949 | S-complete + 2% glucose | Experimental Factor 2 - Media |
MCT_EV2_SD | SA212646 | FL025949 | replicate 2 | Experimental Factor 2 - Replicate |
Collection:
Collection ID: | CO002311 |
Collection Summary: | Metabolomics data were generated by growing the appropriate yeast strains in synthetic complete media supplemented with 2% glucose until they reached saturation (n=3). Cells were then transferred to S-minimal media containing 2% raffinose and leucine and harvested after approximately 8 hours (n=3) at OD600=0.6-0.8. The procedures for metabolite extraction were performed as previously described in [Bricker et al., Science, 2012]. Yeast cultures were pelleted, snap-frozen and kept at −80°C. 5ml of 75% boiled ethanol was added to every frozen pellet. Pellets were vortexed and incubated at 90°C for 5 minutes. All samples were then centrifuged at 5,000 Relative Centrifugal Force (RCF) for 10 minutes. Supernatants were transferred to fresh tubes, evaporated overnight in a Speed Vacuum, and then stored at −80°C until they were run on the mass spectrometer. |
Sample Type: | Yeast cells |
Treatment:
Treatment ID: | TR002330 |
Treatment Summary: | N/A |
Sample Preparation:
Sampleprep ID: | SP002324 |
Sampleprep Summary: | The conditions for liquid chromatography are described in previous studies [Cluntun et al., Cancer Metab., 2015; Lukey et al., Cell Rep., 2019]. Briefly, a hydrophilic interaction liquid chromatography method (HILIC) with an Xbridge amide column (100 × 2.1 mm, 3.5 μm) (Waters) was employed on a Dionex (Ultimate 3000 UHPLC) for compound separation and detection at room temperature. The mobile phase A was 20 mM ammonium acetate and 15 mM ammonium hydroxide in water with 3% acetonitrile, pH 9.0, and the mobile phase B was acetonitrile. The linear gradient was as follows: 0 min, 85% B; 1.5 min, 85% B, 5.5 min, 35% B; 10 min, 35% B, 10.5 min, 35% B, 14.5 min, 35% B, 15 min, 85% B, and 20 min, 85% B. The flow rate was 0.15 ml/min from 0 to 10 min and 15 to 20 min, and 0.3 ml/min from 10.5 to 14.5 min. All solvents were LC-MS grade and purchased from Thermo Fisher Scientific. Mass spectrometry was performed as described in previous studies [Cluntun et al., Cancer Metab., 2015; Lukey et al., Cell Rep., 2019]. Briefly, the Q Exactive MS (Thermo Scientific) is equipped with a heated electrospray ionization probe (HESI), and the relevant parameters are as listed: evaporation temperature, 120°C; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 3.6 kV for positive mode and 2.5 kV for negative mode. Capillary temperature was set at 320°C, and S-lens was 55. A full scan range from 60 to 900 (m/z) was used. The resolution was set at 70,000. The maximum injection time was 200 ms. Automated gain control (AGC) was targeted at 3,000,000 ions. Data were collected, metabolites were identified, and their peak area was recorded using El-MAVEN software [Agrawal et al., Methods Mol. Biol., 2019; Clasquin et al., Curr. Protoc. Bioinformatics, 2012; Melamud et al., Anal. Chem., 2010]. These data were transferred to an Excel spreadsheet (Microsoft, Redmond WA). Metabolite identity was established using a combination of an in-house metabolite library developed using pure purchased standards, the NIST (https://www.nist.gov) and Fiehn libraries [Kind et al, Anal. Chem., 2009]. P-values were derived using a homoscedastic, two-tailed Student's T-test and adjusted using the Benjamini-Hochberg correction procedure. |
Combined analysis:
Analysis ID | AN003642 |
---|---|
Analysis type | MS |
Chromatography type | HILIC |
Chromatography system | Thermo Dionex Ultimate 3000 |
Column | Waters XBridge Amide (100 x 4.6mm,3.5um) |
MS Type | ESI |
MS instrument type | LC-Q Exactive |
MS instrument name | Thermo Q-Exactive |
Ion Mode | UNSPECIFIED |
Units | peak height |
Chromatography:
Chromatography ID: | CH002696 |
Chromatography Summary: | The conditions for liquid chromatography are described in previous studies [Cluntun et al., Cancer Metab., 2015; Lukey et al., Cell Rep., 2019]. Briefly, a hydrophilic interaction liquid chromatography method (HILIC) with an Xbridge amide column (100 × 2.1 mm, 3.5 μm) (Waters) was employed on a Dionex (Ultimate 3000 UHPLC) for compound separation and detection at room temperature. The mobile phase A was 20 mM ammonium acetate and 15 mM ammonium hydroxide in water with 3% acetonitrile, pH 9.0, and the mobile phase B was acetonitrile. The linear gradient was as follows: 0 min, 85% B; 1.5 min, 85% B, 5.5 min, 35% B; 10 min, 35% B, 10.5 min, 35% B, 14.5 min, 35% B, 15 min, 85% B, and 20 min, 85% B. The flow rate was 0.15 ml/min from 0 to 10 min and 15 to 20 min, and 0.3 ml/min from 10.5 to 14.5 min. All solvents were LC-MS grade and purchased from Thermo Fisher Scientific. |
Instrument Name: | Thermo Dionex Ultimate 3000 |
Column Name: | Waters XBridge Amide (100 x 4.6mm,3.5um) |
Flow Gradient: | 0 min, 85% B; 1.5 min, 85% B, 5.5 min, 35% B; 10 min, 35% B, 10.5 min, 35% B, 14.5 min, 35% B, 15 min, 85% B, and 20 min, 85% B |
Flow Rate: | 0.15 ml/min from 0 to 10 min and 15 to 20 min, and 0.3 ml/min from 10.5 to 14.5 min |
Solvent A: | 100% water; 20 mM ammonium acetate; 15 mM ammonium hydroxide; 3% acetonitrile, pH 9.0 |
Solvent B: | 100% acetonitrile |
Chromatography Type: | HILIC |
MS:
MS ID: | MS003393 |
Analysis ID: | AN003642 |
Instrument Name: | Thermo Q-Exactive |
Instrument Type: | LC-Q Exactive |
MS Type: | ESI |
MS Comments: | Data were collected, metabolites were identified, and their peak area was recorded using El-MAVEN software [Agrawal et al., Methods Mol. Biol., 2019; Clasquin et al., Curr. Protoc. Bioinformatics, 2012; Melamud et al., Anal. Chem., 2010]. These data were transferred to an Excel spreadsheet (Microsoft, Redmond WA). Metabolite identity was established using a combination of an in-house metabolite library developed using pure purchased standards, the NIST (https://www.nist.gov) and Fiehn libraries [Kind et al, Anal. Chem., 2009]. P-values were derived using a homoscedastic, two-tailed Student's T-test and adjusted using the Benjamini-Hochberg correction procedure. |
Ion Mode: | UNSPECIFIED |