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MB Sample ID: SA130685
| Local Sample ID: | CP_isoprenaline_3 |
| Subject ID: | SU001623 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001623 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CP_isoprenaline_3 | SA130685 | FL016421 | Isoprenaline_13C-Glucose | Treatment |
Collection:
| Collection ID: | CO001616 |
| Collection Summary: | Cells (9.9x106) were differentiated in 10 cm glass dishes (Corning) coated with 10 mM fibronectin. After 24 hours in culture, cells were treated with 1 µM isoprenaline (or vehicle) for a further 24 hours. Cells were extracted as previously described (14). Briefly, cells were quenched and washed three times with 4 °C Dulbecco’s phosphate buffered saline (DPBS; Invitrogen), then scraped in 750 µL of ice-cold extraction solvent (chloroform: methanol: water = 1:3:1). After scraping, the samples were mixed thoroughly on a vortex mixer for 30 min at 1200 rpm (4 °C) and centrifuged at 20,000 g for 10 min (4 °C). Samples were then evaporated to dryness under a nitrogen stream and frozen at -80 °C until LC-MS analysis. |
| Sample Type: | Macrophages |
Treatment:
| Treatment ID: | TR001636 |
| Treatment Summary: | Differentiated macrophage-like cells (9.9x106) were seeded onto fibronectin-coated glass dishes. After 24 hours in culture, cells were treated with 1 µM isoprenaline (or vehicle) and 11 mM U-13C6-D-Glucose labelled medium (to give a final ratio of 50:50 of U-13C and U-12C D-glucose). |
Sample Preparation:
| Sampleprep ID: | SP001629 |
| Sampleprep Summary: | Cells were extracted as previously described (14). Briefly, cells were quenched and washed three times with 4 °C Dulbecco’s phosphate buffered saline (DPBS; Invitrogen), then scraped in 750 µL of ice-cold extraction solvent (chloroform: methanol: water = 1:3:1). After scraping, the samples were mixed thoroughly on a vortex mixer for 30 min at 1200 rpm (4 °C) and centrifuged at 20,000 g for 10 min (4 °C). Samples were then evaporated to dryness under a nitrogen stream and frozen at -80 °C until LC-MS analysis. |
Combined analysis:
| Analysis ID | AN002576 | AN002577 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
| Column | ZIC-pHILIC (150 x 4.6mm,5um) | ZIC-pHILIC (150 x 4.6mm,5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Intensity | Intensity |
Chromatography:
| Chromatography ID: | CH001886 |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | ZIC-pHILIC (150 x 4.6mm,5um) |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH001887 |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | ZIC-pHILIC (150 x 4.6mm,5um) |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002388 |
| Analysis ID: | AN002576 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometry was performed in polarity switching mode, with the following settings: resolution 35 000, AGC 1x106, m/z range 85-1275, sheath gas 50, auxiliary gas 20, sweep gas 2, probe temperature 150 °C, and capillary temperature 300 °C. For positive ionisation mode the source voltage was set at +4 kV and the S-lens voltage at +50 V. For negative ionisation mode the source voltage was set at -3.5 kV and the S-lens voltage at -50 V. Mass calibration was performed for each polarity before running a metabolomics batch to ensure mass accuracy of < 2 ppm. Approximately 300 authentic metabolite standards were analysed at the start of each batch to provide accurate retention times to facilitate metabolite identification. Metabolomics samples were analysed in random order with periodic injections of the pooled QC, and blank samples, to assess analytical quality and aid downstream metabolite identification procedures. Metabolomics LC-MS Data Processing Raw metabolite data was processed using XCMS (Centwave) software for peak picking and mzMatch.R software for alignment and annotation of related metabolite peaks. Metabolites were then identified using the Excel-based IDEOM software by matching the mass of each peak and its retention time with a database, using a mass accuracy window of 2 ppm and a retention time window of 5% for metabolites matching authentic standards, and 35% for other putative metabolites based on a retention time prediction model (15). Noise and mass spectrometry artefacts were filtered using previously described algorithms (15, 16) to minimise false identifications. Detection of stable isotope labelled metabolite peaks were performed using mzMatch-ISO (17). Initial statistical analysis was performed with IDEOM using peak intensities (height) for all detected putative metabolites. Untargeted multivariate analysis was performed using MetaboAnalyst 4.0 (18), where the complete data sets were also analysed using enrichment analysis and pathway analysis functions (19, 20). Further analysis was undertaken using TraceFinderTM (Thermo) to obtain manually curated accurate peak areas for targeted univariate analyses for metabolites in key pathways. Metabolomics data are presented as fold-change values from 2 individual experiments, each with 4 replicates. Labelled metabolomics data is from a single metabolomics experiment and presented as a mean ± SD. Differences were determined using Student’s t-test where significant interactions were observed. Significance was determined at p values < 0.05. |
| Ion Mode: | POSITIVE |
| MS ID: | MS002389 |
| Analysis ID: | AN002577 |
| Instrument Name: | Thermo Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometry was performed in polarity switching mode, with the following settings: resolution 35 000, AGC 1x106, m/z range 85-1275, sheath gas 50, auxiliary gas 20, sweep gas 2, probe temperature 150 °C, and capillary temperature 300 °C. For positive ionisation mode the source voltage was set at +4 kV and the S-lens voltage at +50 V. For negative ionisation mode the source voltage was set at -3.5 kV and the S-lens voltage at -50 V. Mass calibration was performed for each polarity before running a metabolomics batch to ensure mass accuracy of < 2 ppm. Approximately 300 authentic metabolite standards were analysed at the start of each batch to provide accurate retention times to facilitate metabolite identification. Metabolomics samples were analysed in random order with periodic injections of the pooled QC, and blank samples, to assess analytical quality and aid downstream metabolite identification procedures. Metabolomics LC-MS Data Processing Raw metabolite data was processed using XCMS (Centwave) software for peak picking and mzMatch.R software for alignment and annotation of related metabolite peaks. Metabolites were then identified using the Excel-based IDEOM software by matching the mass of each peak and its retention time with a database, using a mass accuracy window of 2 ppm and a retention time window of 5% for metabolites matching authentic standards, and 35% for other putative metabolites based on a retention time prediction model (15). Noise and mass spectrometry artefacts were filtered using previously described algorithms (15, 16) to minimise false identifications. Detection of stable isotope labelled metabolite peaks were performed using mzMatch-ISO (17). Initial statistical analysis was performed with IDEOM using peak intensities (height) for all detected putative metabolites. Untargeted multivariate analysis was performed using MetaboAnalyst 4.0 (18), where the complete data sets were also analysed using enrichment analysis and pathway analysis functions (19, 20). Further analysis was undertaken using TraceFinderTM (Thermo) to obtain manually curated accurate peak areas for targeted univariate analyses for metabolites in key pathways. Metabolomics data are presented as fold-change values from 2 individual experiments, each with 4 replicates. Labelled metabolomics data is from a single metabolomics experiment and presented as a mean ± SD. Differences were determined using Student’s t-test where significant interactions were observed. Significance was determined at p values < 0.05. |
| Ion Mode: | NEGATIVE |
