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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
Study ID | ST001148 |
Study Title | Effect of cell harvesting technique and storage on metabolic profiles in human skin fibroblasts |
Study Type | Method |
Study Summary | Human 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 |
Department | Neurology |
Last Name | Wilkins |
First Name | Jordan |
Address | 200 First St SW |
wilkins.jordan@mayo.edu | |
Phone | 5072933857 |
Submit Date | 2019-02-27 |
Analysis Type Detail | GC-MS/LC-MS |
Release Date | 2019-09-23 |
Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
mb_sample_id | local_sample_id | Harvest_technique | Storage_time |
---|---|---|---|
SA079794 | 10268_22 | Methanol_dry | 2_weeks |
SA079795 | 10268_23 | Methanol_dry | 2_weeks |
SA079796 | 10268_24 | Methanol_dry | 2_weeks |
SA079797 | 10268_12 | Methanol_dry | 48_hours |
SA079798 | 10268_10 | Methanol_dry | 48_hours |
SA079799 | 10268_11 | Methanol_dry | 48_hours |
SA079800 | 10268_34 | Methanol_dry | 4_weeks |
SA079801 | 10268_36 | Methanol_dry | 4_weeks |
SA079802 | 10268_35 | Methanol_dry | 4_weeks |
SA079785 | 10268_21 | Methanol | 2_weeks |
SA079786 | 10268_20 | Methanol | 2_weeks |
SA079787 | 10268_19 | Methanol | 2_weeks |
SA079788 | 10268_8 | Methanol | 48_hours |
SA079789 | 10268_7 | Methanol | 48_hours |
SA079790 | 10268_9 | Methanol | 48_hours |
SA079791 | 10268_33 | Methanol | 4_weeks |
SA079792 | 10268_32 | Methanol | 4_weeks |
SA079793 | 10268_31 | Methanol | 4_weeks |
SA079803 | 10268_18 | Scraping | 2_weeks |
SA079804 | 10268_17 | Scraping | 2_weeks |
SA079805 | 10268_16 | Scraping | 2_weeks |
SA079806 | 10268_6 | Scraping | 48_hours |
SA079807 | 10268_5 | Scraping | 48_hours |
SA079808 | 10268_4 | Scraping | 48_hours |
SA079809 | 10268_28 | Scraping | 4_weeks |
SA079810 | 10268_30 | Scraping | 4_weeks |
SA079811 | 10268_29 | Scraping | 4_weeks |
SA079812 | 10268_13 | Trypsin | 2_weeks |
SA079813 | 10268_15 | Trypsin | 2_weeks |
SA079814 | 10268_14 | Trypsin | 2_weeks |
SA079815 | 10268_2 | Trypsin | 48_hours |
SA079816 | 10268_3 | Trypsin | 48_hours |
SA079817 | 10268_1 | Trypsin | 48_hours |
SA079818 | 10268_25 | Trypsin | 4_weeks |
SA079819 | 10268_26 | Trypsin | 4_weeks |
SA079820 | 10268_27 | Trypsin | 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) |
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 |