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
Email	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

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

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).

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