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MB Sample ID: SA355193
Local Sample ID: | ctrl_2_1hr |
Subject ID: | SU003402 |
Subject Type: | Cultured cells |
Subject Species: | Bos taurus |
Taxonomy ID: | 9913 |
Select appropriate tab below to view additional metadata details:
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 |
---|---|---|---|---|
ctrl_2_1hr | SA355193 | FL041458 | ctrl_1hr | Treatment |
ctrl_2_1hr | SA355193 | FL041458 | 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. |
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. |
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 |