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/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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Project:

Project ID:PR000767
Project DOI:doi: 10.21228/M8J68Z
Project Title:Effect of harvesting technique and storage on the metabolic profile of patient-derived skin fibroblasts
Project Type:MS targeted metabolomics
Project Summary:Using human skin fibroblasts, we measured the effect of different cell harvesting techniques (trypsinization, scraping, and methanol fixation) as well as storage time on the metabolic profiles of organic acids, amino acids, and acylcarnitines.
Institute:Mayo Clinic
Department:Neurology
Last Name:Wilkins
First Name:Jordan
Address:200 First St SW
Email:wilkins.jordan@mayo.edu
Phone:5072933857

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:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Harvest_technique Storage_time
SA07979410268_22Methanol_dry 2_weeks
SA07979510268_23Methanol_dry 2_weeks
SA07979610268_24Methanol_dry 2_weeks
SA07979710268_12Methanol_dry 48_hours
SA07979810268_10Methanol_dry 48_hours
SA07979910268_11Methanol_dry 48_hours
SA07980010268_34Methanol_dry 4_weeks
SA07980110268_36Methanol_dry 4_weeks
SA07980210268_35Methanol_dry 4_weeks
SA07978510268_21Methanol 2_weeks
SA07978610268_20Methanol 2_weeks
SA07978710268_19Methanol 2_weeks
SA07978810268_8Methanol 48_hours
SA07978910268_7Methanol 48_hours
SA07979010268_9Methanol 48_hours
SA07979110268_33Methanol 4_weeks
SA07979210268_32Methanol 4_weeks
SA07979310268_31Methanol 4_weeks
SA07980310268_18Scraping 2_weeks
SA07980410268_17Scraping 2_weeks
SA07980510268_16Scraping 2_weeks
SA07980610268_6Scraping 48_hours
SA07980710268_5Scraping 48_hours
SA07980810268_4Scraping 48_hours
SA07980910268_28Scraping 4_weeks
SA07981010268_30Scraping 4_weeks
SA07981110268_29Scraping 4_weeks
SA07981210268_13Trypsin 2_weeks
SA07981310268_15Trypsin 2_weeks
SA07981410268_14Trypsin 2_weeks
SA07981510268_2Trypsin 48_hours
SA07981610268_3Trypsin 48_hours
SA07981710268_1Trypsin 48_hours
SA07981810268_25Trypsin 4_weeks
SA07981910268_26Trypsin 4_weeks
SA07982010268_27Trypsin 4_weeks
Showing results 1 to 36 of 36

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)
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
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