Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA079795

Local Sample ID:	10268_23
Subject ID:	SU001213
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	21
Gender:	Male
Cell Biosource Or Supplier:	Mayo Clinic
Cell Strain Details:	10268
Cell Passage Number:	10

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Sample Preparation:

Sampleprep ID:	SP001221
Sampleprep Summary:	Targeted metabolomics analysis of amino acids, acylcarnitines, and metabolites of the TCA cycle was conducted at the Mayo Clinic Metabolomics Center. Samples harvested in methanol were dried using a Speed Vacuum. Cell pellets were sonicated in 100 µL PBS and spiked with 15 - 25 µL of the respective internal standards. Proteins were removed by adding 450 µL of cold 1:1 methanol/acetonitrile solution with subsequent centrifugation for 15 minutes (18,000 x g at 4ºC). Supernatants were transferred to a 1 mL dram and dried under a nitrogen stream for approximately 30 minutes. Prior to detection, TCA cycle metabolites were derivatized using ethoxyamine and then with MtBSTFA + 1% tBDMCS (N-Methyl-N-(t-Butyldimethylsilyl)-Trifluoroacetamide + 1% t-Butyldimethylchlorosilane). Amino acids were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using the Waters AccQ-Fluor Reagent Kit (Cat. # WATO52880). Acylcarnitines were reconstituted in buffer containing 99% MeOH, 1% H2O, 1 mM ammonium formate, and 0.1% formic acid. Organic acids were detected with an Agilent 5977A gas chromatography/mass spectrometry (GC/MS) under electron impact and single ion monitoring conditions. Analytes were separated on an Agilent DB-5MS column (30 m x 0.25 mm x 0.25μm). Sample injection volume was 1 μL performed in splitless mode. The inlet temperature was maintained at 250°C. The carrier gas was helium set at a flow rate of 0.9 ml/min. The initial oven temperature was 120°C set with the following ramp rates: Ramp to 180°C at 25°C/min; Ramp to 270°C at 6°C/min; Ramp to 325°C at 30°C/min. The transfer line temperature was 280°C. Concentrations of lactic acid, fumaric acid, succinic acid, oxaloacetic acid, alpha-ketoglutaric acid, malic acid, 2-hydroxyglutaric acid, cis-aconitic acid, citric acid, and isocitric acid were measured against a calibration curve. Amino acids were analyzed using Thermo TSQ Quantum Ultra mass spectrometer (West Palm Beach, FL) coupled with a Waters ACQUITY ultra performance liquid chromatography BEH C18 column (2.1 mm x 150 mm x. 1.7 μm). Data acquisition was performed using selected reaction monitoring (SRM) and positive electrospray ionization (ESI). Injection volume was 1 μL. The column flow rate was 400 μL/min with an isothermal set at 43°C. Mobile phase A was 1% acetonitrile in 0.1% formic acid, and mobile phase B was 100% acetonitrile. The mass spectrometer was operated with 4000 capillary voltage, 50 sheath gas, 20 auxiliary gas, and 15 sweep gas. The capillary temperature was 2700°C. Collision gas was 1.5 Torr and collision energy was 25 V. The tube lens was kept at 90 V. The concentration of amino acids was calculated against a calibration curve. Acylcarnitines were analyzed using a Waters ACQUITY UPLC system (Milford, MA) coupled with a Thermo TSQ Quantiva tandem mass spectrometer (West Palm Beach, FL) in SRM and positive (H)ESI mode. Analytes were separated on a Waters ACQUITY UPLC BEH C8 column (2.1 mm x 150 mm x. 1.7 μm) with an isothermal temperature of 43°C. The mass spectrometer capillary voltage was set to 4000 with a sheath gas 30, auxiliary gas 5, and sweep gas 2. The ion transfer tube was maintained at 300°C with the vaporizer at 40°C, collision gas at 1.5 Torr, and collision energy at 12 V. Concentrations of carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, octanoylcarnitine, lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine, oleoylcarnitine, and stearoylcarnitine were measured against a calibration curve. Data sets collected by GC/MS were analyzed using Mass Hunter GC/MS Quantitation software version B.07 (Agilent). Analysis of LC/MS data sets was performed using Xcalibur Quant browser (Thermo Scientific).

Sampleprep ID:

SP001221

Sampleprep Summary:

Targeted metabolomics analysis of amino acids, acylcarnitines, and metabolites of the TCA cycle was conducted at the Mayo Clinic Metabolomics Center. Samples harvested in methanol were dried using a Speed Vacuum. Cell pellets were sonicated in 100 µL PBS and spiked with 15 - 25 µL of the respective internal standards. Proteins were removed by adding 450 µL of cold 1:1 methanol/acetonitrile solution with subsequent centrifugation for 15 minutes (18,000 x g at 4ºC). Supernatants were transferred to a 1 mL dram and dried under a nitrogen stream for approximately 30 minutes. Prior to detection, TCA cycle metabolites were derivatized using ethoxyamine and then with MtBSTFA + 1% tBDMCS (N-Methyl-N-(t-Butyldimethylsilyl)-Trifluoroacetamide + 1% t-Butyldimethylchlorosilane). Amino acids were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using the Waters AccQ-Fluor Reagent Kit (Cat. # WATO52880). Acylcarnitines were reconstituted in buffer containing 99% MeOH, 1% H2O, 1 mM ammonium formate, and 0.1% formic acid. Organic acids were detected with an Agilent 5977A gas chromatography/mass spectrometry (GC/MS) under electron impact and single ion monitoring conditions. Analytes were separated on an Agilent DB-5MS column (30 m x 0.25 mm x 0.25μm). Sample injection volume was 1 μL performed in splitless mode. The inlet temperature was maintained at 250°C. The carrier gas was helium set at a flow rate of 0.9 ml/min. The initial oven temperature was 120°C set with the following ramp rates: Ramp to 180°C at 25°C/min; Ramp to 270°C at 6°C/min; Ramp to 325°C at 30°C/min. The transfer line temperature was 280°C. Concentrations of lactic acid, fumaric acid, succinic acid, oxaloacetic acid, alpha-ketoglutaric acid, malic acid, 2-hydroxyglutaric acid, cis-aconitic acid, citric acid, and isocitric acid were measured against a calibration curve. Amino acids were analyzed using Thermo TSQ Quantum Ultra mass spectrometer (West Palm Beach, FL) coupled with a Waters ACQUITY ultra performance liquid chromatography BEH C18 column (2.1 mm x 150 mm x. 1.7 μm). Data acquisition was performed using selected reaction monitoring (SRM) and positive electrospray ionization (ESI). Injection volume was 1 μL. The column flow rate was 400 μL/min with an isothermal set at 43°C. Mobile phase A was 1% acetonitrile in 0.1% formic acid, and mobile phase B was 100% acetonitrile. The mass spectrometer was operated with 4000 capillary voltage, 50 sheath gas, 20 auxiliary gas, and 15 sweep gas. The capillary temperature was 2700°C. Collision gas was 1.5 Torr and collision energy was 25 V. The tube lens was kept at 90 V. The concentration of amino acids was calculated against a calibration curve. Acylcarnitines were analyzed using a Waters ACQUITY UPLC system (Milford, MA) coupled with a Thermo TSQ Quantiva tandem mass spectrometer (West Palm Beach, FL) in SRM and positive (H)ESI mode. Analytes were separated on a Waters ACQUITY UPLC BEH C8 column (2.1 mm x 150 mm x. 1.7 μm) with an isothermal temperature of 43°C. The mass spectrometer capillary voltage was set to 4000 with a sheath gas 30, auxiliary gas 5, and sweep gas 2. The ion transfer tube was maintained at 300°C with the vaporizer at 40°C, collision gas at 1.5 Torr, and collision energy at 12 V. Concentrations of carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, octanoylcarnitine, lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine, oleoylcarnitine, and stearoylcarnitine were measured against a calibration curve. Data sets collected by GC/MS were analyzed using Mass Hunter GC/MS Quantitation software version B.07 (Agilent). Analysis of LC/MS data sets was performed using Xcalibur Quant browser (Thermo Scientific).