Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA355210

Local Sample ID:	gln_1_24hr
Subject ID:	SU003402
Subject Type:	Cultured cells
Subject Species:	Bos taurus
Taxonomy ID:	9913

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Subject:

Subject ID:	SU003402
Subject Type:	Cultured cells
Subject Species:	Bos taurus
Taxonomy ID:	9913

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
gln_1_24hr	SA355210	FL041463	gln_24hr	Treatment
gln_1_24hr	SA355210	FL041463	Bovine aortic endothelial cells	Sample source

Collection:

Collection ID:	CO003395
Collection Summary:	Cell culture media was removed and the cells were rinsed 3x with PBS using an 8-channel pipette. Lysing solution (100µL of 80/20 ACN/H2O)) was added and the cells were put over ice. The wells were flush-mixed 10 times, sonicated for 5 minutes, then centrifuged at 3,400 rpm for 10 minutes at 10ºC. The supernatant was then transferred to a 96-well plate containing 200µg of pre-aliquoted tag in each well.
Sample Type:	Endothelial cells

Treatment:

Treatment ID:	TR003411
Treatment Summary:	Bulk bovine aortic endothelial cells were cultured in complete media. These bulk plates were used to seed 96-well plates at 40,000 cells per well in 200µL of media. These cultures were maintained for 24 hours in complete media supplemented with 10% FBS before nutrient deprivation. Triplicate controls were included at every time point and were media swapped to complete media + 10% dialyzed FBS. Triplicate 96-well plates were used for nutrient deprivation experiments, and a separate plate was cultured for cell counting using a hemocytometer following manufacturer instructions. Tags were allocated to different wells to reduce bias caused by tag interactions. A single row (B) was left empty during culture. These wells were used to create a calibration curve for each analyte centered around the approximate biological concentration in controls.

Treatment ID:

TR003411

Treatment Summary:

Bulk bovine aortic endothelial cells were cultured in complete media. These bulk plates were used to seed 96-well plates at 40,000 cells per well in 200µL of media. These cultures were maintained for 24 hours in complete media supplemented with 10% FBS before nutrient deprivation. Triplicate controls were included at every time point and were media swapped to complete media + 10% dialyzed FBS. Triplicate 96-well plates were used for nutrient deprivation experiments, and a separate plate was cultured for cell counting using a hemocytometer following manufacturer instructions. Tags were allocated to different wells to reduce bias caused by tag interactions. A single row (B) was left empty during culture. These wells were used to create a calibration curve for each analyte centered around the approximate biological concentration in controls.

Sample Preparation:

Sampleprep ID:	SP003409
Sampleprep Summary:	DMTMM mediated coupling of amine tags to biological acids is well studied and previous reports were used as a basis for acid tagging. Briefly, the coupling of biological acids to the amine tag was initiated with the addition of 25µL of a DMTMM/NMM solution (75mM DMTMM, 100mM NMM in 95/5 ACN/H2O)). The 96-well plate was allowed to react for 30 minutes at room temperature before another addition (12.5µL) of DMTMM/NMM to ensure reaction completion and account for water-mediated DMTMM degradation. After 4 hours total reaction time, 50µL of 50mM myristic acid was added and allowed to react for an additional 30 minutes to quench the reaction. All 96 well lysates are mixed into a 15mL falcon tube after the reaction is quenched. This reaction solution is mixed 1:1 with chloroform, vortexed for 30s, then centrifuged at 2,000 rpm for 5 minutes to ensure phase separation. The top aqueous layer is then taken for WCX enrichment. Supelclean 500 mg WCX columns were used to selectively enrich quaternary amine-tagged metabolites. Columns were conditioned with 4 mL of 90/10 (MeOH/H2O) and then equilibrated with 4 mL of loading buffer (10% MeOH in 20mM ammonium formate pH 8.5). The extracted reaction solution was mixed 1:1 with loading buffer before injection. The column was then rinsed with 4 mL of loading buffer, followed by elution with 8 mL of 90/10 (MeOH/H2O) + 1% formic acid. While singly tagged analytes readily elute at mL 2-3 under these conditions, doubly tagged analytes require more fractions to be collected. The first 1mL of this elution was discarded and the rest were pooled, dried, and reconstituted into 50µL of 25/25/50 (MeOH/H2O/ACN) to ensure solubility of all tagged analytes.

Sampleprep ID:

SP003409

Sampleprep Summary:

DMTMM mediated coupling of amine tags to biological acids is well studied and previous reports were used as a basis for acid tagging. Briefly, the coupling of biological acids to the amine tag was initiated with the addition of 25µL of a DMTMM/NMM solution (75mM DMTMM, 100mM NMM in 95/5 ACN/H2O)). The 96-well plate was allowed to react for 30 minutes at room temperature before another addition (12.5µL) of DMTMM/NMM to ensure reaction completion and account for water-mediated DMTMM degradation. After 4 hours total reaction time, 50µL of 50mM myristic acid was added and allowed to react for an additional 30 minutes to quench the reaction. All 96 well lysates are mixed into a 15mL falcon tube after the reaction is quenched. This reaction solution is mixed 1:1 with chloroform, vortexed for 30s, then centrifuged at 2,000 rpm for 5 minutes to ensure phase separation. The top aqueous layer is then taken for WCX enrichment. Supelclean 500 mg WCX columns were used to selectively enrich quaternary amine-tagged metabolites. Columns were conditioned with 4 mL of 90/10 (MeOH/H2O) and then equilibrated with 4 mL of loading buffer (10% MeOH in 20mM ammonium formate pH 8.5). The extracted reaction solution was mixed 1:1 with loading buffer before injection. The column was then rinsed with 4 mL of loading buffer, followed by elution with 8 mL of 90/10 (MeOH/H2O) + 1% formic acid. While singly tagged analytes readily elute at mL 2-3 under these conditions, doubly tagged analytes require more fractions to be collected. The first 1mL of this elution was discarded and the rest were pooled, dried, and reconstituted into 50µL of 25/25/50 (MeOH/H2O/ACN) to ensure solubility of all tagged analytes.

Combined analysis:

Analysis ID	AN005377
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Orbitrap ID-X tribrid
Ion Mode	POSITIVE
Units	nM

Chromatography:

Chromatography ID:	CH004076
Instrument Name:	Thermo Vanquish
Column Name:	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:	50ºC
Flow Gradient:	0–1 min, 100% B; 6 min, 45% B; 8.5 min, 45% B; 8.6 – 18 min, 0% B
Flow Rate:	300µL/min
Solvent A:	90% Water, 10% Acetonitrile
Solvent B:	95% Acetonitrile, 5% Water
Chromatography Type:	HILIC

MS:

MS ID:	MS005106
Analysis ID:	AN005377
Instrument Name:	Thermo Orbitrap ID-X tribrid
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The IDX was operated with an isolation width of 12 m/z, maximum injection time of 502 ms, AGC of 1e6, positive spray voltage of 3.5 kV, sheath gas of 50 arbitrary units (Arb), aux gas of 10 Arb, sweep gas of 1 Arb, a vaporizer temperature of 350ºC, and a capillary temperature of 325ºC.
Ion Mode:	POSITIVE