Summary of Study ST003282

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 PR002034. The data can be accessed directly via it's Project DOI: 10.21228/M8PV5M This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003282
Study Title	96-plex metabolomics studies on nutrient-deprived endothelial cells
Study Summary	A NeuCode tag which allows for cost efficient incorporation of isotopes is used to enable a 96-plex assay for small molecule metabolomics. A 96-well plate of cell lysates is analyzed simultaneously, providing absolute quantitation of 38 metabolites across 96 samples in a single LC-MS injection.
Institute	Saint Louis University
Last Name	Armbruster
First Name	Michael
Address	4448 Swiss Stone Ln E Apt 3C, Ypsilanti, Michigan, 48197, USA
Email	armbrustermr@gmail.com
Phone	3145501620
Submit Date	2024-02-15
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-07-15
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002034
Project DOI:	doi: 10.21228/M8PV5M
Project Title:	High Throughput Metabolomics using 96-plex Isotope Tagging
Project Summary:	Chemical tags with 96 unique masses are reported which alleviate the metabolomic workflow bottleneck and allow for absolute quantitation. A metabolic screen for carboxylic acids was performed on mammalian cells deprived of various nutrients and showed 24% RSD and analysis of 288 samples in 2 hours.
Institute:	Saint Louis University
Department:	Chemistry
Last Name:	Armbruster
First Name:	Michael
Address:	3501 Laclede Ave, St Louis, MO 63103
Email:	armbrustermr@gmail.com
Phone:	3145501620
Funding Source:	5R01GM134081

Subject:

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

Factors:

Subject type: Cultured cells; Subject species: Bos taurus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment	Sample source
SA355193	ctrl_2_1hr	ctrl_1hr	Bovine aortic endothelial cells
SA355194	ctrl_1_1hr	ctrl_1hr	Bovine aortic endothelial cells
SA355195	ctrl_3_1hr	ctrl_1hr	Bovine aortic endothelial cells
SA355196	ctrl_3_24hr	ctrl_24hr	Bovine aortic endothelial cells
SA355197	ctrl_2_24hr	ctrl_24hr	Bovine aortic endothelial cells
SA355198	ctrl_1_24hr	ctrl_24hr	Bovine aortic endothelial cells
SA355199	ctrl_1_4hr	ctrl_4hr	Bovine aortic endothelial cells
SA355200	ctrl_3_4hr	ctrl_4hr	Bovine aortic endothelial cells
SA355201	ctrl_2_4hr	ctrl_4hr	Bovine aortic endothelial cells
SA355202	ctrl_1_8hr	ctrl_8hr	Bovine aortic endothelial cells
SA355203	ctrl_2_8hr	ctrl_8hr	Bovine aortic endothelial cells
SA355204	ctrl_3_8hr	ctrl_8hr	Bovine aortic endothelial cells
SA355205	gln_1_1hr	gln_1hr	Bovine aortic endothelial cells
SA355206	gln_3_1hr	gln_1hr	Bovine aortic endothelial cells
SA355207	gln_2_1hr	gln_1hr	Bovine aortic endothelial cells
SA355208	gln_3_24hr	gln_24hr	Bovine aortic endothelial cells
SA355209	gln_2_24hr	gln_24hr	Bovine aortic endothelial cells
SA355210	gln_1_24hr	gln_24hr	Bovine aortic endothelial cells
SA355211	gln_3_4hr	gln_4hr	Bovine aortic endothelial cells
SA355212	gln_1_4hr	gln_4hr	Bovine aortic endothelial cells
SA355213	gln_2_4hr	gln_4hr	Bovine aortic endothelial cells
SA355214	gln_3_8hr	gln_8hr	Bovine aortic endothelial cells
SA355215	gln_2_8hr	gln_8hr	Bovine aortic endothelial cells
SA355216	gln_1_8hr	gln_8hr	Bovine aortic endothelial cells
SA355217	gln_gluc_3_1hr	gln_gluc_1hr	Bovine aortic endothelial cells
SA355218	gln_gluc_2_1hr	gln_gluc_1hr	Bovine aortic endothelial cells
SA355219	gln_gluc_1_1hr	gln_gluc_1hr	Bovine aortic endothelial cells
SA355220	gln_gluc_2_24hr	gln_gluc_24hr	Bovine aortic endothelial cells
SA355221	gln_gluc_1_24hr	gln_gluc_24hr	Bovine aortic endothelial cells
SA355222	gln_gluc_3_24hr	gln_gluc_24hr	Bovine aortic endothelial cells
SA355223	gln_gluc_1_4hr	gln_gluc_4hr	Bovine aortic endothelial cells
SA355224	gln_gluc_2_4hr	gln_gluc_4hr	Bovine aortic endothelial cells
SA355225	gln_gluc_3_4hr	gln_gluc_4hr	Bovine aortic endothelial cells
SA355226	gln_gluc_3_8hr	gln_gluc_8hr	Bovine aortic endothelial cells
SA355227	gln_gluc_2_8hr	gln_gluc_8hr	Bovine aortic endothelial cells
SA355228	gln_gluc_1_8hr	gln_gluc_8hr	Bovine aortic endothelial cells
SA355229	gln_pyr_1_1hr	gln_pyr_1hr	Bovine aortic endothelial cells
SA355230	gln_pyr_3_1hr	gln_pyr_1hr	Bovine aortic endothelial cells
SA355231	gln_pyr_2_1hr	gln_pyr_1hr	Bovine aortic endothelial cells
SA355232	gln_pyr_3_24hr	gln_pyr_24hr	Bovine aortic endothelial cells
SA355233	gln_pyr_1_24hr	gln_pyr_24hr	Bovine aortic endothelial cells
SA355234	gln_pyr_2_24hr	gln_pyr_24hr	Bovine aortic endothelial cells
SA355235	gln_pyr_3_4hr	gln_pyr_4hr	Bovine aortic endothelial cells
SA355236	gln_pyr_2_4hr	gln_pyr_4hr	Bovine aortic endothelial cells
SA355237	gln_pyr_1_4hr	gln_pyr_4hr	Bovine aortic endothelial cells
SA355238	gln_pyr_3_8hr	gln_pyr_8hr	Bovine aortic endothelial cells
SA355239	gln_pyr_2_8hr	gln_pyr_8hr	Bovine aortic endothelial cells
SA355240	gln_pyr_1_8hr	gln_pyr_8hr	Bovine aortic endothelial cells
SA355241	gluc_3_1hr	gluc_1hr	Bovine aortic endothelial cells
SA355242	gluc_1_1hr	gluc_1hr	Bovine aortic endothelial cells
SA355243	gluc_2_1hr	gluc_1hr	Bovine aortic endothelial cells
SA355244	gluc_1_24hr	gluc_24hr	Bovine aortic endothelial cells
SA355245	gluc_2_24hr	gluc_24hr	Bovine aortic endothelial cells
SA355246	gluc_3_24hr	gluc_24hr	Bovine aortic endothelial cells
SA355247	gluc_1_4hr	gluc_4hr	Bovine aortic endothelial cells
SA355248	gluc_2_4hr	gluc_4hr	Bovine aortic endothelial cells
SA355249	gluc_3_4hr	gluc_4hr	Bovine aortic endothelial cells
SA355250	gluc_3_8hr	gluc_8hr	Bovine aortic endothelial cells
SA355251	gluc_1_8hr	gluc_8hr	Bovine aortic endothelial cells
SA355252	gluc_2_8hr	gluc_8hr	Bovine aortic endothelial cells
SA355253	gluc_pyr_2_1hr	gluc_pyr_1hr	Bovine aortic endothelial cells
SA355254	gluc_pyr_1_1hr	gluc_pyr_1hr	Bovine aortic endothelial cells
SA355255	gluc_pyr_3_1hr	gluc_pyr_1hr	Bovine aortic endothelial cells
SA355256	gluc_pyr_3_24hr	gluc_pyr_24hr	Bovine aortic endothelial cells
SA355257	gluc_pyr_2_24hr	gluc_pyr_24hr	Bovine aortic endothelial cells
SA355258	gluc_pyr_1_24hr	gluc_pyr_24hr	Bovine aortic endothelial cells
SA355259	gluc_pyr_1_4hr	gluc_pyr_4hr	Bovine aortic endothelial cells
SA355260	gluc_pyr_3_4hr	gluc_pyr_4hr	Bovine aortic endothelial cells
SA355261	gluc_pyr_2_4hr	gluc_pyr_4hr	Bovine aortic endothelial cells
SA355262	gluc_pyr_2_8hr	gluc_pyr_8hr	Bovine aortic endothelial cells
SA355263	gluc_pyr_3_8hr	gluc_pyr_8hr	Bovine aortic endothelial cells
SA355264	gluc_pyr_1_8hr	gluc_pyr_8hr	Bovine aortic endothelial cells
SA355265	pyr_1_1hr	pyr_1hr	Bovine aortic endothelial cells
SA355266	pyr_3_1hr	pyr_1hr	Bovine aortic endothelial cells
SA355267	pyr_2_1hr	pyr_1hr	Bovine aortic endothelial cells
SA355268	pyr_3_24hr	pyr_24hr	Bovine aortic endothelial cells
SA355269	pyr_1_24hr	pyr_24hr	Bovine aortic endothelial cells
SA355270	pyr_2_24hr	pyr_24hr	Bovine aortic endothelial cells
SA355271	pyr_3_4hr	pyr_4hr	Bovine aortic endothelial cells
SA355272	pyr_2_4hr	pyr_4hr	Bovine aortic endothelial cells
SA355273	pyr_1_4hr	pyr_4hr	Bovine aortic endothelial cells
SA355274	pyr_3_8hr	pyr_8hr	Bovine aortic endothelial cells
SA355275	pyr_2_8hr	pyr_8hr	Bovine aortic endothelial cells
SA355276	pyr_1_8hr	pyr_8hr	Bovine aortic endothelial cells

Showing results 1 to 84 of 84

Showing results 1 to 84 of 84

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