Summary of Study ST001148

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000767. The data can be accessed directly via it's Project DOI: 10.21228/M8J68Z This work is supported by NIH grant, U2C- DK119886.

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Study IDST001148
Study TitleEffect of cell harvesting technique and storage on metabolic profiles in human skin fibroblasts
Study TypeMethod
Study SummaryHuman skin fibroblasts were cultured in MEM media supplemented with 15% FBS. Cells were harvested using (i) trypsinization, (ii) scraping, (iii) methanol fixation and scraping, (iV) methanol fixation, scraping, and drying. Targeted metabolomics analysis of organic acids, amino acids, and acycarnitines was conducted at Mayo Clinic Metabolomics Core Facility.
Institute
Mayo Clinic
DepartmentNeurology
Last NameWilkins
First NameJordan
Address200 First St SW
Emailwilkins.jordan@mayo.edu
Phone5072933857
Submit Date2019-02-27
Analysis Type DetailGC-MS/LC-MS
Release Date2019-09-23
Release Version1
Jordan Wilkins Jordan Wilkins
https://dx.doi.org/10.21228/M8J68Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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
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