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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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_A3_SD	SA212641	FL025944	BY4743	Strain
MCT_A3_SD	SA212641	FL025944	mct1 deletion	Genotype
MCT_A3_SD	SA212641	FL025944	CTP1-overexpression vector	Experimental Factor 1 - Vector
MCT_A3_SD	SA212641	FL025944	S-complete + 2% glucose	Experimental Factor 2 - Media
MCT_A3_SD	SA212641	FL025944	replicate 3	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.

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	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:	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