Summary of Study ST003858
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 PR002415. The data can be accessed directly via it's Project DOI: 10.21228/M8GC1D 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 | ST003858 |
| Study Title | Metabolomics study on Xenopus tropicalis tadpoles overexpressing human IMPDH2 pathogenic variant |
| Study Summary | Xenopus tropicalis embryos were injected with 1 ng of mRNA encoding either WT human IMPDH2 or a gain-of-function variant of human IMPDH2, S160del, at the two-cell stage and raised until stage 41. Whole tadpoles lysates were analyzed by LC-MS to assess the consequences of IMPDH2 dysregulation on purine nucleotide metabolism. We find that overexpression of WT human IMPDH2 does not significantly alter purine nucleotide metabolism of the developing tadpole, but S160del overexpression significantly increases the levels of guanine nucleotides and uric acid, a degredation product of purine nucleotides. These results suggest increased activity through the guanine nucleotide biosynthetic pathway caused by IMPDH2 hyperactivity. |
| Institute | University of Washington |
| Department | Biochemistry |
| Laboratory | Wills |
| Last Name | O'Neill |
| First Name | Audrey |
| Address | 1959 NE Pacific St., Seattle, WA, 98195, USA |
| aoneill1@uw.edu | |
| Phone | 206-543-2866 |
| Submit Date | 2025-04-08 |
| Num Groups | 3 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-04-23 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002415 |
| Project DOI: | doi: 10.21228/M8GC1D |
| Project Title: | IMPDH2 dysregulation in disease |
| Project Summary: | Targeted metabolomics of Xenopus tropicalis tadpoles to study gain-of-function disease mutation in IMPDH2 |
| Institute: | University of Washington |
| Department: | Biochemistry |
| Laboratory: | Wills |
| Last Name: | O'Neill |
| First Name: | Audrey |
| Address: | 1959 NE Pacific St., Seattle, WA, 98195, USA |
| Email: | aoneill1@uw.edu |
| Phone: | 206-543-2866 |
| Funding Source: | R35GM149542 from NIGMS, R01GM148490 from NIGMS, R01NS099124 from NINDS |
Subject:
| Subject ID: | SU003992 |
| Subject Type: | Amphibian |
| Subject Species: | Xenopus tropicalis |
| Taxonomy ID: | 8364 |
| Age Or Age Range: | Stage 41 |
Factors:
Subject type: Amphibian; Subject species: Xenopus tropicalis (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Condition |
|---|---|---|---|
| SA424389 | 2024-11-25_Wills-15-Blank_NEG_001 | Blank | - |
| SA424390 | 2024-11-25_Wills-15-PrepBlank | Blank | - |
| SA424391 | 2024-11-25_Wills-15-Blank_POS_005 | Blank | - |
| SA424392 | 2024-11-25_Wills-15-Blank_POS_004 | Blank | - |
| SA424393 | 2024-11-25_Wills-15-Blank_POS_003 | Blank | - |
| SA424394 | 2024-11-25_Wills-15-Blank_POS_002 | Blank | - |
| SA424395 | 2024-11-25_Wills-15-Blank_POS_001 | Blank | - |
| SA424396 | 2024-11-25_Wills-15-Blank_NEG_005 | Blank | - |
| SA424397 | 2024-11-25_Wills-15-Blank_NEG_004 | Blank | - |
| SA424398 | 2024-11-25_Wills-15-Blank_NEG_003 | Blank | - |
| SA424399 | 2024-11-25_Wills-15-Blank_NEG_002 | Blank | - |
| SA424400 | 2024-11-25_Wills-15-QC(I)#1 | QC | - |
| SA424401 | 2024-11-25_Wills-15-QC(S)#3 | QC | - |
| SA424402 | 2024-11-25_Wills-15-QC(S)#2 | QC | - |
| SA424403 | 2024-11-25_Wills-15-QC(S)#1 | QC | - |
| SA424404 | 2024-11-25_Wills-15-QC(I)#3 | QC | - |
| SA424405 | 2024-11-25_Wills-15-QC(I)#2 | QC | - |
| SA424406 | 2024-11-25_Wills-15-13 | Whole_tadpole | S160del-injected |
| SA424407 | 2024-11-25_Wills-15-6 | Whole_tadpole | S160del-injected |
| SA424408 | 2024-11-25_Wills-15-9 | Whole_tadpole | S160del-injected |
| SA424409 | 2024-11-25_Wills-15-10 | Whole_tadpole | S160del-injected |
| SA424410 | 2024-11-25_Wills-15-2 | Whole_tadpole | S160del-injected |
| SA424411 | 2024-11-25_Wills-15-8 | Whole_tadpole | Uninjected |
| SA424412 | 2024-11-25_Wills-15-14 | Whole_tadpole | Uninjected |
| SA424413 | 2024-11-25_Wills-15-4 | Whole_tadpole | Uninjected |
| SA424414 | 2024-11-25_Wills-15-3 | Whole_tadpole | Uninjected |
| SA424415 | 2024-11-25_Wills-15-12 | Whole_tadpole | Uninjected |
| SA424416 | 2024-11-25_Wills-15-7 | Whole_tadpole | WT-injected |
| SA424417 | 2024-11-25_Wills-15-5 | Whole_tadpole | WT-injected |
| SA424418 | 2024-11-25_Wills-15-11 | Whole_tadpole | WT-injected |
| SA424419 | 2024-11-25_Wills-15-15 | Whole_tadpole | WT-injected |
| SA424420 | 2024-11-25_Wills-15-1 | Whole_tadpole | WT-injected |
| Showing results 1 to 32 of 32 |
Collection:
| Collection ID: | CO003985 |
| Collection Summary: | For each aggregate tissue sample, 10 whole tadpoles were euthanized with a lethal dose of MS-222 at St. 41 and transferred into tubes. Media was removed, and samples were immediately flash frozen in liquid nitrogen. Five aggregate tissue samples were collected per group. |
| Collection Protocol Filename: | 2024-11-25_Wills-15_methods.pdf |
| Sample Type: | Whole tadpoles |
Treatment:
| Treatment ID: | TR004001 |
| Treatment Summary: | 1 ng of mRNA encoding either human WT IMPDH2 or S160del IMPDH2 was injected into Xenopus tropicalis embryos at the two-cell stage. A subset of embryos collected from the same clutch were left uninjected and set aside as a control group. Tadpoles were reared to Stage 41 in 1/9x MR (embryo rearing media). |
Sample Preparation:
| Sampleprep ID: | SP003998 |
| Sampleprep Summary: | Aqueous metabolites for targeted LC-MS profiling of 15 aggregate tissue samples were extracted using a protein precipitation method similar to the one previously described (Kurup et al., Aging 2021; Patel et al., Cell Reports 2022) by the Northwest Metabolomics Research Center. Samples were first homogenized in 200 µL purified deionized water at 4 ˚C, and then 800 µL of cold methanol containing 124 µM 6C13-glucose and 25.9 µM 2C13-glutamate was added (reference internal standards were added to the samples in order to monitor sample prep). 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 (left-over protein pallet was used for BCA assay). Lastly, recovered supernatants were dried on a SpeedVac and reconstituted in 0.5 mL of LC-matching solvent containing 17.8 µM 2C13-tyrosine and 39.2 3C13-lactate (reference internal standards were added to the reconstituting solvent in order to monitor LC-MS performance). Samples were transferred into LC vials and placed into a temperature controlled autosampler kept at 4 °C for LC-MS analysis. |
| Sampleprep Protocol Filename: | 2024-11-25_Wills-15_methods.pdf |
Combined analysis:
| Analysis ID | AN006339 | AN006340 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Shimadzu Nexera X2 | Shimadzu Nexera X2 |
| Column | Waters XBridge Premier BEH Amide (150 x 2.1mm, 2.5um) | Waters XBridge Premier BEH Amide (150 x 2.1mm, 2.5um) |
| MS Type | ESI | ESI |
| MS instrument type | Triple quadrupole | Triple quadrupole |
| MS instrument name | ABI Sciex Triple Quad 6500+ | ABI Sciex Triple Quad 6500+ |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Peak intensity | Peak intensity |
Chromatography:
| Chromatography ID: | CH004810 |
| Chromatography Summary: | Chromatography for positive ionization mode: Targeted LC-MS metabolite analysis was performed with a similar protocol as previously described by the Northwest Metabolomics Research Center on a duplex-LC-MS system composed of two Shimadzu UPLC pumps, CTC Analytics PAL HTC-xt temperature-controlled auto-sampler and AB Sciex 6500+ Triple Quadrupole MS equipped with ESI ionization source (Kurup et al., Aging 2021; Patel et al., Cell Reports 2022). UPLC pumps were connected to the auto-sampler in parallel and were able to perform two chromatography separations independently from each other. Each sample was injected twice on two identical analytical columns (Waters XBridge Premier BEH Amide column, Part # 186009930) performing separations in hydrophilic interaction liquid chromatography (HILIC) mode. While one column was performing separation and MS data acquisition in ESI+ ionization mode, the other column was getting equilibrated for sample injection, chromatography separation and MS data acquisition in ESI- mode. Each chromatography separation was 16 minutes (total analysis time per sample was 32 minutes). |
| Methods Filename: | 2024-11-25_Wills-15_methods.pdf |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters XBridge Premier BEH Amide (150 x 2.1mm, 2.5um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-3 min: 95% B, 3-8 min: 95%-50% B, 8-12 min: 50% B, 12-13 min: 50%-95% B, 13-18 min: 95% B |
| Flow Rate: | 0.3 mL/min |
| Solvent A: | 95%H2O/ 3% acetonitrile/ 2% methanol + 0.2% acetic acid; 10 mM ammonium acetate |
| Solvent B: | 93%acetonitrile/ 5% H2O/ 2% methanol + 0.2% acetic acid; 10 mM ammonium acetate |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH004811 |
| Chromatography Summary: | Chromatography for negative ionization mode: Targeted LC-MS metabolite analysis was performed with a similar protocol as previously described by the Northwest Metabolomics Research Center on a duplex-LC-MS system composed of two Shimadzu UPLC pumps, CTC Analytics PAL HTC-xt temperature-controlled auto-sampler and AB Sciex 6500+ Triple Quadrupole MS equipped with ESI ionization source (Kurup et al., Aging 2021; Patel et al., Cell Reports 2022). UPLC pumps were connected to the auto-sampler in parallel and were able to perform two chromatography separations independently from each other. Each sample was injected twice on two identical analytical columns (Waters XBridge Premier BEH Amide column, Part # 186009930) performing separations in hydrophilic interaction liquid chromatography (HILIC) mode. While one column was performing separation and MS data acquisition in ESI+ ionization mode, the other column was getting equilibrated for sample injection, chromatography separation and MS data acquisition in ESI- mode. Each chromatography separation was 16 minutes (total analysis time per sample was 32 minutes). |
| Methods Filename: | 2024-11-25_Wills-15_methods.pdf |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters XBridge Premier BEH Amide (150 x 2.1mm, 2.5um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-1.5 min: 95% B, 1.5-6 min: 95%-70% B, 6-10 min: 70% B, 10-12 min: 70%-45% B, 12-14 min: 45% B, 14-15 min: 45%-95% B, 15-18 min: 95% B |
| Flow Rate: | 0.3 mL/min |
| Solvent A: | 95%H2O/ 5% methanol + 0.3% acetic acid; 10 mM ammonium acetate |
| Solvent B: | 90%acetonitrile/ 5% H2O/ 5% methanol + 0.3% acetic acid; 10 mM ammonium acetate |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS006040 |
| Analysis ID: | AN006339 |
| Instrument Name: | ABI Sciex Triple Quad 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 373 metabolites (plus 4 spiked reference internal standards). Up to 175 metabolites (plus 4 spiked standards) were measured across the study set, and over 95% of measured metabolites were measured across all the samples. In addition to the study samples, two sets of quality control (QC) samples were used to monitor the assay performance as well as data reproducibility. One QC [QC(I)] was a pooled human serum sample used to monitor system performance. The other QC [QC(S)] was pooled study samples, and this QC was used to monitor data reproducibility. Each QC sample was injected per every 10 study samples. The data were highly reproducible with a median CV of 3.9 %. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | 2024-11-25_Wills-15_methods.pdf |
| MS ID: | MS006041 |
| Analysis ID: | AN006340 |
| Instrument Name: | ABI Sciex Triple Quad 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 373 metabolites (plus 4 spiked reference internal standards). Up to 175 metabolites (plus 4 spiked standards) were measured across the study set, and over 95% of measured metabolites were measured across all the samples. In addition to the study samples, two sets of quality control (QC) samples were used to monitor the assay performance as well as data reproducibility. One QC [QC(I)] was a pooled human serum sample used to monitor system performance. The other QC [QC(S)] was pooled study samples, and this QC was used to monitor data reproducibility. Each QC sample was injected per every 10 study samples. The data were highly reproducible with a median CV of 3.9 %. |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | 2024-11-25_Wills-15_methods.pdf |
