Summary of Study ST003685
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 PR002285. The data can be accessed directly via it's Project DOI: 10.21228/M88G2Z 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.
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| Study ID | ST003685 |
| Study Title | Investigation of C. elegans adah-1 gene for potential adenosine deaminase activity via quantification of predicted substrate and product in control animals and animals with knockdown in expression of adah-1 |
| Study Summary | We have previously demonstrated that the C. elegans pnp-1 gene encodes a purine nucleoside phosphorylase that catalyzes the conversion of inosine to hypoxanthine. We hypothesize that the C. elegans adah-1 (ADA homolog) gene encodes the adenosine deaminase that acts upstream of PNP-1 to produce inosine from adenosine. To investigate the hypothesis that ADAH-1 functions as a canonical adenosine deaminase, we used RNAi to knockdown expression of ADAH-1, and used LC-MS to compare relative levels of adenosine, inosine, and hypoxanthine in the knockdown animals and an RNAi control (L4440). As a control we also confirmed the activity of PNP-1, by comparing levels of adenosine, inosine, and hypoxanthine in control (N2) and the loss of function mutant pnp-1(jy90). As expected we found that levels of hypoxanthine were reduced and levels of inosine were increased in pnp-1 mutants relative to control. In contrast, RNAi against adah-1 led to significantly increased levels of adenosine, and decreased levels of inosine and the downstream metabolite hypoxanthine. We conclude that ADAH-1 is a bona fide adenosine deaminase. |
| Institute | Pennsylvania State University |
| Last Name | Hanna-Rose |
| First Name | Wendy |
| Address | 432 Science Drive, LSB Room 104D |
| wxh21@psu.edu | |
| Phone | 8149337569 |
| Submit Date | 2025-01-16 |
| Num Groups | 4 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-02-14 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002285 |
| Project DOI: | doi: 10.21228/M88G2Z |
| Project Title: | Adenosine Deaminase function in C. elegans |
| Project Summary: | We have previously demonstrated that the C. elegans pnp-1 gene encodes a purine nucleoside phosphorylase that catalyzes the conversion of inosine to hypoxanthine. We hypothesize that the C. elegans adah-1 gene encodes the adenosine deaminase that acts upstream of PNP-1 to produce inosine from adenosine. To investigate the hypothesis that ADAH-1 functions as a canonical adenosine deaminase, we used RNAi to knockdown expression of ADAH-1, and used LC-MS to compare relative levels of adenosine, inosine, and hypoxanthine in the knockdown animals and an RNAi control (L4440). As a control we also confirmed the activity of pnp-1, by comparing levels of adenosine, inosine, and hypoxanthine in control (N2) and the loss of function mutant pnp-1(jy90). As expected we found that levels of hypoxanthine were reduced and levels of inosine were increased in pnp-1 mutants relative to control. In contrast, RNAi against adah-1 led to significantly increased levels of adenosine, and decreased levels of inosine and the downstream metabolite hypoxanthine. We conclude that ADAH-1 is a bona fide adenosine deaminase. |
| Institute: | Pennsylvania State University |
| Department: | Biochemistry and Molecular Biology |
| Last Name: | Hanna-Rose |
| First Name: | Wendy |
| Address: | 432 Science Drive, LSB Room 104D |
| Email: | wxh21@psu.edu |
| Phone: | 8149337569 |
Subject:
| Subject ID: | SU003817 |
| Subject Type: | Invertebrate |
| Subject Species: | Caenorhabditis elegans |
| Taxonomy ID: | 6239 |
| Genotype Strain: | N2, pnp-1(jy90) |
| Age Or Age Range: | adult |
Factors:
Subject type: Invertebrate; Subject species: Caenorhabditis elegans (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Sample_class |
|---|---|---|---|
| SA402435 | adah-1 RNAi 6 | Worms | adah-1(RNAi) |
| SA402436 | adah-1 RNAi 4 | Worms | adah-1(RNAi) |
| SA402437 | adah-1 RNAi 1 | Worms | adah-1(RNAi) |
| SA402438 | adah-1 RNAi 7 | Worms | adah-1(RNAi) |
| SA402439 | adah-1 RNAi 8 | Worms | adah-1(RNAi) |
| SA402440 | adah-1 RNAi 3 | Worms | adah-1(RNAi) |
| SA402441 | adah-1 RNAi 5 | Worms | adah-1(RNAi) |
| SA402442 | adah-1 RNAi 2 | Worms | adah-1(RNAi) |
| SA402416 | Blank 2 | Worms | Blank |
| SA402417 | Blank 1 | Worms | Blank |
| SA402418 | Blank 3 | Worms | Blank |
| SA402419 | L4440 7 | Worms | L4440 RNAi |
| SA402420 | L4440 4 | Worms | L4440 RNAi |
| SA402421 | L4440 8 | Worms | L4440 RNAi |
| SA402422 | L4440 1 | Worms | L4440 RNAi |
| SA402423 | L440 6 | Worms | L4440 RNAi |
| SA402424 | L4440 3 | Worms | L4440 RNAi |
| SA402425 | L4440 2 | Worms | L4440 RNAi |
| SA402426 | L4440 5 | Worms | L4440 RNAi |
| SA402427 | N2 6 | Worms | N2 |
| SA402428 | N2 7 | Worms | N2 |
| SA402429 | N2 1 | Worms | N2 |
| SA402430 | N2 8 | Worms | N2 |
| SA402431 | N2 3 | Worms | N2 |
| SA402432 | N2 4 | Worms | N2 |
| SA402433 | N2 5 | Worms | N2 |
| SA402434 | N2 2 | Worms | N2 |
| SA402443 | pnp-1 7 | Worms | pnp-1(jy90) |
| SA402444 | pnp-1 5 | Worms | pnp-1(jy90) |
| SA402445 | pnp-1 2 | Worms | pnp-1(jy90) |
| SA402446 | pnp-1 6 | Worms | pnp-1(jy90) |
| SA402447 | pnp-1 3 | Worms | pnp-1(jy90) |
| SA402448 | pnp-1 4 | Worms | pnp-1(jy90) |
| SA402449 | pnp-1 1 | Worms | pnp-1(jy90) |
| SA402450 | pnp-1 8 | Worms | pnp-1(jy90) |
| SA402451 | Pooled Sample 3 | Worms | pooled sample |
| SA402452 | Pooled Sample 1 | Worms | pooled sample |
| SA402453 | Pooled Sample 2 | Worms | pooled sample |
| Showing results 1 to 38 of 38 |
Collection:
| Collection ID: | CO003810 |
| Collection Summary: | For adah-1 RNAi analysis, six L4 (fourth larval) stage N2 (lab control strain) worms were picked onto 10 cm RNAi plates seeded with L4440 (empty vector RNAi) or adah-1(RNAi) in E. coli strain HT115 overnight cultures grown at 37 °C and incubated at 20°C. Gravid adult progeny were synchronized and 1500-2000 eggs were plated onto 10 cm RNAi plates seeded with the respective RNAi and incubated at 20°C for ~70 hours. Six 10 cm RNAi plates were used for each condition per experiment. For analysis of the pnp-1 mutant, synchronized N2 or pnp-1(jy90) L1 (first larval stage)larvae were seeded onto 10 cm plates and kept at 20 oC 4 days until they became adults, which were harvested, washed with M9 buffer, and transferred to 1.5 mL reaction tubes. Approximately 100 µL of packed worms were collected and flash frozen in liquid nitrogen immediately. The samples were kept at -80oC until shipping. |
| Sample Type: | Worms |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003826 |
| Treatment Summary: | We analyzed eight biological replicates of synchronized adult N2 control, pnp-1(jy90), RNAi control and adah-1A(RNAi) animals. |
Sample Preparation:
| Sampleprep ID: | SP003824 |
| Sampleprep Summary: | Samples were extracted in 1 ml of 3:3:2 acetonitrile/isopropyl alcohol/H2O with 1 μm chlorpropamide as internal standard. Samples were homogenized using a PrecellysTM 24 homogenizer. Extracts from samples were dried under vacuum, resuspended in HPLC Optima Water (Thermo Fisher Scientific, Waltham, MA) |
Combined analysis:
| Analysis ID | AN006048 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Supelco Titan C18 (100 x 2.1mm,1.9um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Exactive Plus Orbitrap |
| Ion Mode | NEGATIVE |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH004596 |
| Chromatography Summary: | Samples were separated on a Supelco (Bellefonte, PA) Titan C18 column (100 × _2.1 mm 1.9 μm particle size) using a water-methanol gradient with tributylamine added to the aqueous mobile phase. The LC-MS platform consisted of Dionex Ultimate 3000 quaternary HPLC pump, 3000 column compartment, 3000 autosampler, and an Exactive plus orbitrap mass spectrometer controlled by Xcalibur 2.2 software (all from ThermoFisher Scientific, San Jose, CA). The HPLC column was maintained at 30 ◦C and a flow rate of 200 uL/min. Solvent A was 3% aqueous methanol with 10 mM tributylamine and 15 mM acetic acid; solvent B was methanol. The gradient was 0 min., 0% B; 5 min., 20% B; 7.5 min., 20% B; 13 min., 55% B; 15.5 min., 95% B, 18.5 min., 95% B; 19 min., 0% B; 25 min 0% B. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Supelco Titan C18 (100 x 2.1mm,1.9um) |
| Column Temperature: | 30 °C |
| Flow Gradient: | 0 min., 0% B; 5 min., 20% B; 7.5 min., 20% B; 13 min., 55% B; 15.5 min., 95% B, 18.5 min., 95% B; 19 min., 0% B; 25 min 0% |
| Flow Rate: | 200 uL/min |
| Solvent A: | 3% methanol/97% water; 10 mM tributylamine; 15 mM acetic acid |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005757 |
| Analysis ID: | AN006048 |
| Instrument Name: | Thermo Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The orbitrap was operated in negative ion mode at maximum resolution (140,000) and scanned from m/z 85 to m/z 1000 |
| Ion Mode: | NEGATIVE |
