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MB Sample ID: SA217741
| Local Sample ID: | DHEA3 |
| Subject ID: | SU002356 |
| Subject Type: | Other organism |
| Subject Species: | Xenopus tropicalis |
| Age Or Age Range: | Stage 41-43 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002356 |
| Subject Type: | Other organism |
| Subject Species: | Xenopus tropicalis |
| Age Or Age Range: | Stage 41-43 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| DHEA3 | SA217741 | FL026429 | DHEA | Condition |
Collection:
| Collection ID: | CO002349 |
| Collection Summary: | Tadpoles were anesthetized with 0.05% MS-222 in 1/9x MR and tested for response to touch prior to tissue collection. Injections of miniRuby or 4nmol of 2DG were performed or tadpoles were incubated in media with DMSO, DHEA, or g6pdi/ 10 whole animals were collected at 24 hours post treatment, media was removed, and samples were frozen on dry ice immediately. |
| Sample Type: | Stage 43 tadpoles |
Treatment:
| Treatment ID: | TR002368 |
| Treatment Summary: | Dihydroepiandrosterone (BioVision, 2172) was resuspended to a 1M stock in DMSO and g6pdi (Cayman Chemical Co., 31484) to a 10mM stock in DMSO. Tadples were reared with 0.1% DMSO, 25µM DHEA or 10µM g6pdi diluted in 1/9x MR until collection after a 24 treatment. For 2DG injections, wild type tadpoles at stage 41 were anesthetized with MS-222 and moved from culture dish to a thickly coasted agarose dish in one drop of media. Excess media was removed. Using a microinjector, a pulled needle containing vMO and labeled dextran tracer was inserted into the ventral tail vein and 2×2 nL of 1M 2DG (Sigma, D8375) were injected. Controls for 2DG experiments were injected with equal volumes of tracer. Embryos were returned to fresh media and screened for tracer fluorescence in the bloodstream. Injected animals were kept in 1/9x MR until collection after a 24 treatment. |
Sample Preparation:
| Sampleprep ID: | SP002362 |
| Sampleprep Summary: | Aqueous metabolites for targeted LC-MS analysis were extracted using a protein precipitation method similar to the one described elsewhere (Mathon et al., 2019; Meador et al., 2020). Samples were first homogenized in 200 µL purified deionized water at 4 ˚C, and then 800 µL of methanol containing 6C13-glucose and 2C13-glutamate (reference internal standards) was added. Afterwards samples were vortexed, stored for 30 minutes at -20 ˚C, sonicated in an ice bath for 10 minutes, centrifuged for 15 min at 14,000 rpm and 4 ˚C, and then 600 µL of supernatant was collected from each sample. Lastly, recovered supernatants were dried on a SpeedVac and reconstituted in 1.0 mL of LC-matching solvent containing 2C13-tyrosine and 3C13-lactate (reference internal standards). |
| Sampleprep Protocol Filename: | JPatel_LC-MS_Methods.pdf |
Combined analysis:
| Analysis ID | AN003710 | AN003711 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Shimadzu Nexera X2 | Shimadzu Nexera X2 |
| Column | Waters XBridge BEH Amide XP | Waters XBridge BEH Amide XP |
| MS Type | ESI | ESI |
| MS instrument type | Triple quadrupole | Triple quadrupole |
| MS instrument name | ABI Sciex 6500+ | ABI Sciex 6500+ |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Peak Intensity | Peak intensity |
Chromatography:
| Chromatography ID: | CH002749 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters XBridge BEH Amide XP |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003459 |
| Analysis ID: | AN003710 |
| Instrument Name: | ABI Sciex 6500+ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | MS data acquisition was performed in multiple-reaction-monitoring (MRM) mode. LC-MS system was controlled using AB Sciex Analyst 1.6.3 software. Measured MS peaks were integrated using AB Sciex MultiQuant 3.0.3 software. The LC-MS assay was targeting 361 metabolites (plus 4 spiked reference internal standards). |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | JPatel_LC-MS_Methods.pdf |
| MS ID: | MS003460 |
| Analysis ID: | AN003711 |
| Instrument Name: | ABI Sciex 6500+ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | MS data acquisition was performed in multiple-reaction-monitoring (MRM) mode. LC-MS system was controlled using AB Sciex Analyst 1.6.3 software. Measured MS peaks were integrated using AB Sciex MultiQuant 3.0.3 software. The LC-MS assay was targeting 361 metabolites (plus 4 spiked reference internal standards). |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | JPatel_LC-MS_Methods.pdf |
