Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA212641

Local Sample ID:	MCT_A3_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)

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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.

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.