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MB Sample ID: SA300850
| Local Sample ID: | 62122_c |
| Subject ID: | SU002912 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 63 - 73 |
| Gender: | Male and female |
| Species Group: | Mammals |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002912 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 63 - 73 |
| Gender: | Male and female |
| Species Group: | Mammals |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 62122_c | SA300850 | FL036545 | M | Sex |
| 62122_c | SA300850 | FL036545 | Control | Treatment |
Collection:
| Collection ID: | CO002905 |
| Collection Summary: | Metabolomics of conditioned media was performed at the Beth Israel Deaconess Medical Center Mass Spectrometry Core according to published protocols (M. Yuan, S. B. Breitkopf, X. Yang, J. M. Asara, A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nat Protoc 7, 872-881 (2012)). hVCF (2 x 106) were seeded and grown to confluence and treated with IL-1β (10 ng/ml) for 24h. Conditioned media (1ml) from the vehicle or IL-1β treated hVCF cells were used for the metabolomics analysis. |
| Sample Type: | Cultured fibroblasts |
Treatment:
| Treatment ID: | TR002921 |
| Treatment Summary: | hVCF (2 x 106) were seeded and grown to confluence and treated with IL-1β (10 ng/ml) for 24h. Conditioned media (1ml) from the vehicle or IL-1β treated hVCF cells were used for the metabolomics analysis. |
Sample Preparation:
| Sampleprep ID: | SP002918 |
| Sampleprep Summary: | 500 μL of chilled -80°C 80% methanol was added to 15ml tubes containing 1ml of conditioned media and evaporated using Speedvac to pellet the metabolites. SRM with polarity switching with a QTRAP 5500 mass spectrometer (AB/SCIEX) was used to assay 300 polar molecules, based on the previously published protocol. |
Combined analysis:
| Analysis ID | AN004561 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Shimadzu Prominence UFLC HPLC |
| Column | Waters XBridge Amide (100 x 4.6mm,3.5um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | ABI Sciex 6500 QTrap |
| Ion Mode | UNSPECIFIED |
| Units | Q3 Peak Area (area under curve) |
Chromatography:
| Chromatography ID: | CH003427 |
| Methods Filename: | nprot_2012_024_v2.pdf |
| Instrument Name: | Shimadzu Prominence UFLC HPLC |
| Column Name: | Waters XBridge Amide (100 x 4.6mm,3.5um) |
| Column Temperature: | 475 |
| Flow Gradient: | Gradients were run with a Prominence UFLC HPLC (Shimadzu) starting from 85% buffer B (HPLC grade acetonitrile) to 42% B from 0-5 minutes; 42% B to 0% B from 5-16 minutes; 0% B was held from 16-24 minutes; 0% B to 85% B from 24-25 minutes; 85% B was held for 7 minutes to re-equilibrate the column. |
| Flow Rate: | 400uL/min |
| Solvent A: | 20 mM ammonium hydroxide/20 mM ammonium acetate (pH=9.0) in 95:5 water:acetonitrile |
| Solvent B: | HPLC grade acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004307 |
| Analysis ID: | AN004561 |
| Instrument Name: | ABI Sciex 6500 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Ion Mode Positive/Negative Switching: Metabolomics of conditioned media was performed at the Beth Israel Deaconess Medical Center Mass Spectrometry Core according to published protocols (M. Yuan, S. B. Breitkopf, X. Yang, J. M. Asara, A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nat Protoc 7, 872-881 (2012)). hVCF (2 x 106) were seeded and grown to confluence and treated with IL-1β (10 ng/ml) for 24h. Conditioned media (1ml) from the vehicle or IL-1β treated hVCF cells were used for the metabolomics analysis. 500 μL of chilled -80°C 80% methanol was added to 15ml tubes containing 1ml of conditioned media and evaporated using Speedvac to pellet the metabolites. Samples were re-suspended using 20 mL HPLC grade water for mass spectrometry. 5-7 μL were injected and analyzed using a hybrid 6500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu) via selected reaction monitoring (SRM) of a total of 283 endogenous water soluble metabolites for steady-state analyses of samples. Some metabolites were targeted in both positive and negative ion mode for a total of 304 SRM transitions using positive/negative ion polarity switching. ESI voltage was +4950V in positive ion mode and –4500V in negative ion mode. The dwell time was 3 ms per SRM transition and the total cycle time was 1.39 seconds. Approximately 10-14 data points were acquired per detected metabolite. Samples were delivered to the mass spectrometer via hydrophilic interaction chromatography (HILIC) using a 4.6 mm i.d x 10 cm Amide XBridge column (Waters) at 400 μL/min. Gradients were run with a Prominence UFLC HPLC (Shimadzu) starting from 85% buffer B (HPLC grade acetonitrile) to 42% B from 0-5 minutes; 42% B to 0% B from 5-16 minutes; 0% B was held from 16-24 minutes; 0% B to 85% B from 24-25 minutes; 85% B was held for 7 minutes to re-equilibrate the column. Buffer A was comprised of 20 mM ammonium hydroxide/20 mM ammonium acetate (pH=9.0) in 95:5 water:acetonitrile. Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v3.0 software (AB/SCIEX) |
| Ion Mode: | UNSPECIFIED |
| Analysis Protocol File: | nprot_2012_024_v2.pdf |
