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MB Sample ID: SA079793
Local Sample ID: | 10268_31 |
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 |
Select appropriate tab below to view additional metadata details:
Subject:
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 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
10268_31 | SA079793 | FL012220 | Methanol | Harvest_technique |
10268_31 | SA079793 | FL012220 | 4_weeks | Storage_time |
Collection:
Collection ID: | CO001207 |
Collection Summary: | Prior to harvest, media was removed and cells were rinsed with PBS. For trypsinization, cells were dissociated by incubation with 0.05% trypsin at 37ºC for 5 minutes. Dissociated cells were resuspended in MEM (containing 15% FBS) to neutralize trypsin activity. Cells were centrifuged at 1000 x g for 5 min at 4ºC. Pelleted cells were resuspended in PBS and centrifuged at 1000 x g for 5 min at 4°C. The supernatant was aspirated, cell pellets were flash frozen in liquid nitrogen, and frozen pellets were stored at -80ºC. For scraping, cells were detached in 1 mL PBS using a rubber scraper. Detached cells were centrifuged at 1000 x g for 5 min at 4°C, and PBS was aspirated. Cell pellets were flash frozen in liquid nitrogen and stored at -80ºC. For methanol fixation, 1 mL 80% methanol (pre-chilled on dry ice) was added to cells. Tissue culture plates were rocked by hand for about 20 seconds to evenly distribute methanol solution. Cells were detached in 80% methanol using a rubber scraper. Suspensions were transferred to an Eppendorf tube and stored at -80ºC. For methanol fixation and drying, cells were harvested in 1 mL 80% methanol, and the supernatant was evaporated using a Speed Vacuum at room temperature. Dried samples were stored at -80ºC. Frozen samples were stored at -80ºC for 48 hours, two weeks, or four weeks. |
Sample Type: | Cultured fibroblasts |
Collection Frequency: | 48 hours, 2 weeks, and 4 weeks |
Storage Conditions: | -80℃ |
Storage Vials: | 1.5 mL Eppendorf tubes |
Treatment:
Treatment ID: | TR001228 |
Treatment Summary: | Collection method and storage time were variable as described in collection summary |
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). |
Combined analysis:
Analysis ID | AN001893 | AN001894 | AN001895 |
---|---|---|---|
Analysis type | MS | MS | MS |
Chromatography type | GC | Reversed phase | Reversed phase |
Chromatography system | Agilent 7890B | Waters Acquity | Waters Acquity |
Column | Agilent HP5-MS (30m x 0.25mm, 0.25 um) | Waters Acquity BEH C18 (150 x 2mm,1.7um) | Waters Acquity BEH C8 (150 x 2.1mm,1.7um) |
MS Type | EI | ESI | ESI |
MS instrument type | Single quadrupole | Triple quadrupole | Triple quadrupole |
MS instrument name | Agilent 5977A | Thermo Quantiva | Thermo Quantum Ultra |
Ion Mode | POSITIVE | POSITIVE | POSITIVE |
Units | micromol metabolite per gram protein | micromol metabolite per gram protein | micromol metabolite per gram protein |
Chromatography:
Chromatography ID: | CH001370 |
Chromatography Summary: | 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. |
Instrument Name: | Agilent 7890B |
Column Name: | Agilent HP5-MS (30m x 0.25mm, 0.25 um) |
Chromatography Type: | GC |
Chromatography ID: | CH001371 |
Chromatography Summary: | 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. |
Instrument Name: | Waters Acquity |
Column Name: | Waters Acquity BEH C18 (150 x 2mm,1.7um) |
Column Temperature: | 43 |
Flow Rate: | 400ul/min |
Solvent A: | 99% water/1% acetonitrile; 0.1% formic acid |
Solvent B: | 100% acetonitrile |
Chromatography Type: | Reversed phase |
Chromatography ID: | CH001372 |
Chromatography Summary: | 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. |
Instrument Name: | Waters Acquity |
Column Name: | Waters Acquity BEH C8 (150 x 2.1mm,1.7um) |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS001749 |
Analysis ID: | AN001893 |
Instrument Name: | Agilent 5977A |
Instrument Type: | Single quadrupole |
MS Type: | EI |
MS Comments: | Mass Hunter acquisition B.07, Mass Hunter Quantitation B.08 (Agilent) |
Ion Mode: | POSITIVE |
MS ID: | MS001750 |
Analysis ID: | AN001894 |
Instrument Name: | Thermo Quantiva |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | Xcalibur acquisition and Quantitation 4.0.27 (Thermo Electron) |
Ion Mode: | POSITIVE |
MS ID: | MS001751 |
Analysis ID: | AN001895 |
Instrument Name: | Thermo Quantum Ultra |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | Xcalibur acquisition and Quantitation 2.0.7 (Thermo Electron) |
Ion Mode: | POSITIVE |