#METABOLOMICS WORKBENCH HannaRose_20250116_072502 DATATRACK_ID:5535 STUDY_ID:ST003685 ANALYSIS_ID:AN006048 PROJECT_ID:PR002285 VERSION 1 CREATED_ON 01-27-2025 #PROJECT PR:PROJECT_TITLE Adenosine Deaminase function in C. elegans PR:PROJECT_SUMMARY We have previously demonstrated that the C. elegans pnp-1 gene encodes a purine PR:PROJECT_SUMMARY nucleoside phosphorylase that catalyzes the conversion of inosine to PR:PROJECT_SUMMARY hypoxanthine. We hypothesize that the C. elegans adah-1 gene encodes the PR:PROJECT_SUMMARY adenosine deaminase that acts upstream of PNP-1 to produce inosine from PR:PROJECT_SUMMARY adenosine. To investigate the hypothesis that ADAH-1 functions as a canonical PR:PROJECT_SUMMARY adenosine deaminase, we used RNAi to knockdown expression of ADAH-1, and used PR:PROJECT_SUMMARY LC-MS to compare relative levels of adenosine, inosine, and hypoxanthine in the PR:PROJECT_SUMMARY knockdown animals and an RNAi control (L4440). As a control we also confirmed PR:PROJECT_SUMMARY the activity of pnp-1, by comparing levels of adenosine, inosine, and PR:PROJECT_SUMMARY hypoxanthine in control (N2) and the loss of function mutant pnp-1(jy90). As PR:PROJECT_SUMMARY expected we found that levels of hypoxanthine were reduced and levels of inosine PR:PROJECT_SUMMARY were increased in pnp-1 mutants relative to control. In contrast, RNAi against PR:PROJECT_SUMMARY adah-1 led to significantly increased levels of adenosine, and decreased levels PR:PROJECT_SUMMARY of inosine and the downstream metabolite hypoxanthine. We conclude that ADAH-1 PR:PROJECT_SUMMARY is a bona fide adenosine deaminase. PR:INSTITUTE Pennsylvania State University PR:DEPARTMENT Biochemistry and Molecular Biology PR:LAST_NAME Hanna-Rose PR:FIRST_NAME Wendy PR:ADDRESS 432 Science Drive, LSB Room 104D PR:EMAIL wxh21@psu.edu PR:PHONE 8149337569 PR:DOI http://dx.doi.org/10.21228/M88G2Z #STUDY ST:STUDY_TITLE Investigation of C. elegans adah-1 gene for potential adenosine deaminase ST:STUDY_TITLE activity via quantification of predicted substrate and product in control ST:STUDY_TITLE animals and animals with knockdown in expression of adah-1 ST:STUDY_SUMMARY We have previously demonstrated that the C. elegans pnp-1 gene encodes a purine ST:STUDY_SUMMARY nucleoside phosphorylase that catalyzes the conversion of inosine to ST:STUDY_SUMMARY hypoxanthine. We hypothesize that the C. elegans adah-1 (ADA homolog) gene ST:STUDY_SUMMARY encodes the adenosine deaminase that acts upstream of PNP-1 to produce inosine ST:STUDY_SUMMARY from adenosine. To investigate the hypothesis that ADAH-1 functions as a ST:STUDY_SUMMARY canonical adenosine deaminase, we used RNAi to knockdown expression of ADAH-1, ST:STUDY_SUMMARY and used LC-MS to compare relative levels of adenosine, inosine, and ST:STUDY_SUMMARY hypoxanthine in the knockdown animals and an RNAi control (L4440). As a control ST:STUDY_SUMMARY we also confirmed the activity of PNP-1, by comparing levels of adenosine, ST:STUDY_SUMMARY inosine, and hypoxanthine in control (N2) and the loss of function mutant ST:STUDY_SUMMARY pnp-1(jy90). As expected we found that levels of hypoxanthine were reduced and ST:STUDY_SUMMARY levels of inosine were increased in pnp-1 mutants relative to control. In ST:STUDY_SUMMARY contrast, RNAi against adah-1 led to significantly increased levels of ST:STUDY_SUMMARY adenosine, and decreased levels of inosine and the downstream metabolite ST:STUDY_SUMMARY hypoxanthine. We conclude that ADAH-1 is a bona fide adenosine deaminase. ST:INSTITUTE Pennsylvania State University ST:LAST_NAME Hanna-Rose ST:FIRST_NAME Wendy ST:ADDRESS 432 Science Drive, LSB Room 104D ST:EMAIL wxh21@psu.edu ST:PHONE 8149337569 ST:SUBMIT_DATE 2025-01-16 #SUBJECT SU:SUBJECT_TYPE Invertebrate SU:SUBJECT_SPECIES Caenorhabditis elegans SU:TAXONOMY_ID 6239 SU:GENOTYPE_STRAIN N2, pnp-1(jy90) SU:AGE_OR_AGE_RANGE adult #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - adah-1 RNAi 1 Sample source:Worms | Sample_class:adah-1(RNAi) RAW_FILE_NAME=1111202151.mzML SUBJECT_SAMPLE_FACTORS - adah-1 RNAi 2 Sample source:Worms | Sample_class:adah-1(RNAi) RAW_FILE_NAME=1111202147.mzML SUBJECT_SAMPLE_FACTORS - adah-1 RNAi 3 Sample source:Worms | Sample_class:adah-1(RNAi) RAW_FILE_NAME=1111202137.mzML SUBJECT_SAMPLE_FACTORS - adah-1 RNAi 4 Sample source:Worms | Sample_class:adah-1(RNAi) RAW_FILE_NAME=1111202123.mzML SUBJECT_SAMPLE_FACTORS - adah-1 RNAi 5 Sample source:Worms | Sample_class:adah-1(RNAi) RAW_FILE_NAME=1111202141.mzML SUBJECT_SAMPLE_FACTORS - adah-1 RNAi 6 Sample source:Worms | Sample_class:adah-1(RNAi) RAW_FILE_NAME=1111202113.mzML SUBJECT_SAMPLE_FACTORS - adah-1 RNAi 7 Sample source:Worms | Sample_class:adah-1(RNAi) RAW_FILE_NAME=1111202118.mzML SUBJECT_SAMPLE_FACTORS - adah-1 RNAi 8 Sample source:Worms | Sample_class:adah-1(RNAi) RAW_FILE_NAME=1111202108.mzML SUBJECT_SAMPLE_FACTORS - Blank 1 Sample source:Worms | Sample_class:Blank RAW_FILE_NAME=1111202149.mzML SUBJECT_SAMPLE_FACTORS - Blank 2 Sample source:Worms | Sample_class:Blank RAW_FILE_NAME=1111202127.mzML SUBJECT_SAMPLE_FACTORS - Blank 3 Sample source:Worms | Sample_class:Blank RAW_FILE_NAME=1111202101.mzML SUBJECT_SAMPLE_FACTORS - L440 6 Sample source:Worms | Sample_class:L4440 RNAi RAW_FILE_NAME=1111202138.mzML SUBJECT_SAMPLE_FACTORS - L4440 1 Sample source:Worms | Sample_class:L4440 RNAi RAW_FILE_NAME=1111202134.mzML SUBJECT_SAMPLE_FACTORS - L4440 2 Sample source:Worms | Sample_class:L4440 RNAi RAW_FILE_NAME=1111202145.mzML SUBJECT_SAMPLE_FACTORS - L4440 3 Sample source:Worms | Sample_class:L4440 RNAi RAW_FILE_NAME=1111202142.mzML SUBJECT_SAMPLE_FACTORS - L4440 4 Sample source:Worms | Sample_class:L4440 RNAi RAW_FILE_NAME=1111202111.mzML SUBJECT_SAMPLE_FACTORS - L4440 5 Sample source:Worms | Sample_class:L4440 RNAi RAW_FILE_NAME=1111202148.mzML SUBJECT_SAMPLE_FACTORS - L4440 7 Sample source:Worms | Sample_class:L4440 RNAi RAW_FILE_NAME=1111202119.mzML SUBJECT_SAMPLE_FACTORS - L4440 8 Sample source:Worms | Sample_class:L4440 RNAi RAW_FILE_NAME=1111202126.mzML SUBJECT_SAMPLE_FACTORS - N2 1 Sample source:Worms | Sample_class:N2 RAW_FILE_NAME=1111202121.mzML SUBJECT_SAMPLE_FACTORS - N2 2 Sample source:Worms | Sample_class:N2 RAW_FILE_NAME=1111202104.mzML SUBJECT_SAMPLE_FACTORS - N2 3 Sample source:Worms | Sample_class:N2 RAW_FILE_NAME=1111202140.mzML SUBJECT_SAMPLE_FACTORS - N2 4 Sample source:Worms | Sample_class:N2 RAW_FILE_NAME=1111202112.mzML SUBJECT_SAMPLE_FACTORS - N2 5 Sample source:Worms | Sample_class:N2 RAW_FILE_NAME=1111202144.mzML SUBJECT_SAMPLE_FACTORS - N2 6 Sample source:Worms | Sample_class:N2 RAW_FILE_NAME=1111202130.mzML SUBJECT_SAMPLE_FACTORS - N2 7 Sample source:Worms | Sample_class:N2 RAW_FILE_NAME=1111202124.mzML SUBJECT_SAMPLE_FACTORS - N2 8 Sample source:Worms | Sample_class:N2 RAW_FILE_NAME=1111202116.mzML SUBJECT_SAMPLE_FACTORS - pnp-1 1 Sample source:Worms | Sample_class:pnp-1(jy90) RAW_FILE_NAME=1111202154.mzML SUBJECT_SAMPLE_FACTORS - pnp-1 2 Sample source:Worms | Sample_class:pnp-1(jy90) RAW_FILE_NAME=1111202129.mzML SUBJECT_SAMPLE_FACTORS - pnp-1 3 Sample source:Worms | Sample_class:pnp-1(jy90) RAW_FILE_NAME=1111202146.mzML SUBJECT_SAMPLE_FACTORS - pnp-1 4 Sample source:Worms | Sample_class:pnp-1(jy90) RAW_FILE_NAME=1111202152.mzML SUBJECT_SAMPLE_FACTORS - pnp-1 5 Sample source:Worms | Sample_class:pnp-1(jy90) RAW_FILE_NAME=1111202107.mzML SUBJECT_SAMPLE_FACTORS - pnp-1 6 Sample source:Worms | Sample_class:pnp-1(jy90) RAW_FILE_NAME=1111202132.mzML SUBJECT_SAMPLE_FACTORS - pnp-1 7 Sample source:Worms | Sample_class:pnp-1(jy90) RAW_FILE_NAME=1111202106.mzML SUBJECT_SAMPLE_FACTORS - pnp-1 8 Sample source:Worms | Sample_class:pnp-1(jy90) RAW_FILE_NAME=1111202117.mzML SUBJECT_SAMPLE_FACTORS - Pooled Sample 1 Sample source:Worms | Sample_class:pooled sample RAW_FILE_NAME=1111202110.mzML SUBJECT_SAMPLE_FACTORS - Pooled Sample 2 Sample source:Worms | Sample_class:pooled sample RAW_FILE_NAME=1111202128.mzML SUBJECT_SAMPLE_FACTORS - Pooled Sample 3 Sample source:Worms | Sample_class:pooled sample RAW_FILE_NAME=1111202102.mzML #COLLECTION CO:COLLECTION_SUMMARY For adah-1 RNAi analysis, six L4 (fourth larval) stage N2 (lab control strain) CO:COLLECTION_SUMMARY worms were picked onto 10 cm RNAi plates seeded with L4440 (empty vector RNAi) CO:COLLECTION_SUMMARY or adah-1(RNAi) in E. coli strain HT115 overnight cultures grown at 37 °C and CO:COLLECTION_SUMMARY incubated at 20°C. Gravid adult progeny were synchronized and 1500-2000 eggs CO:COLLECTION_SUMMARY were plated onto 10 cm RNAi plates seeded with the respective RNAi and incubated CO:COLLECTION_SUMMARY at 20°C for ~70 hours. Six 10 cm RNAi plates were used for each condition per CO:COLLECTION_SUMMARY experiment. For analysis of the pnp-1 mutant, synchronized N2 or pnp-1(jy90) L1 CO:COLLECTION_SUMMARY (first larval stage)larvae were seeded onto 10 cm plates and kept at 20 oC 4 CO:COLLECTION_SUMMARY days until they became adults, which were harvested, washed with M9 buffer, and CO:COLLECTION_SUMMARY transferred to 1.5 mL reaction tubes. Approximately 100 µL of packed worms were CO:COLLECTION_SUMMARY collected and flash frozen in liquid nitrogen immediately. The samples were kept CO:COLLECTION_SUMMARY at -80oC until shipping. CO:SAMPLE_TYPE Worms CO:STORAGE_CONDITIONS -80â #TREATMENT TR:TREATMENT_SUMMARY We analyzed eight biological replicates of synchronized adult N2 control, TR:TREATMENT_SUMMARY pnp-1(jy90), RNAi control and adah-1A(RNAi) animals. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were extracted in 1 ml of 3:3:2 acetonitrile/isopropyl alcohol/H2O with SP:SAMPLEPREP_SUMMARY 1 μm chlorpropamide as internal standard. Samples were homogenized using a SP:SAMPLEPREP_SUMMARY PrecellysTM 24 homogenizer. Extracts from samples were dried under vacuum, SP:SAMPLEPREP_SUMMARY resuspended in HPLC Optima Water (Thermo Fisher Scientific, Waltham, MA) #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were separated on a Supelco (Bellefonte, PA) Titan C18 column (100 à CH:CHROMATOGRAPHY_SUMMARY _2.1 mm 1.9 μm particle size) using a water-methanol gradient with CH:CHROMATOGRAPHY_SUMMARY tributylamine added to the aqueous mobile phase. The LC-MS platform consisted of CH:CHROMATOGRAPHY_SUMMARY Dionex Ultimate 3000 quaternary HPLC pump, 3000 column compartment, 3000 CH:CHROMATOGRAPHY_SUMMARY autosampler, and an Exactive plus orbitrap mass spectrometer controlled by CH:CHROMATOGRAPHY_SUMMARY Xcalibur 2.2 software (all from ThermoFisher Scientific, San Jose, CA). The HPLC CH:CHROMATOGRAPHY_SUMMARY column was maintained at 30 â¦C and a flow rate of 200 uL/min. Solvent A was 3% CH:CHROMATOGRAPHY_SUMMARY aqueous methanol with 10 mM tributylamine and 15 mM acetic acid; solvent B was CH:CHROMATOGRAPHY_SUMMARY methanol. The gradient was 0 min., 0% B; 5 min., 20% B; 7.5 min., 20% B; 13 CH:CHROMATOGRAPHY_SUMMARY min., 55% B; 15.5 min., 95% B, 18.5 min., 95% B; 19 min., 0% B; 25 min 0% B. CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Supelco Titan C18 (100 x 2.1mm,1.9um) CH:COLUMN_TEMPERATURE 30 °C CH:FLOW_GRADIENT 0 min., 0% B; 5 min., 20% B; 7.5 min., 20% B; 13 min., 55% B; 15.5 min., 95% B, CH:FLOW_GRADIENT 18.5 min., 95% B; 19 min., 0% B; 25 min 0% CH:FLOW_RATE 200 uL/min CH:SOLVENT_A 3% methanol/97% water; 10 mM tributylamine; 15 mM acetic acid CH:SOLVENT_B 100% methanol CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS The orbitrap was operated in negative ion mode at maximum resolution (140,000) MS:MS_COMMENTS and scanned from m/z 85 to m/z 1000 MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples adah-1 RNAi 1 adah-1 RNAi 2 adah-1 RNAi 3 adah-1 RNAi 4 adah-1 RNAi 5 adah-1 RNAi 6 adah-1 RNAi 7 adah-1 RNAi 8 L440 6 L4440 1 L4440 2 L4440 3 L4440 4 L4440 5 L4440 7 L4440 8 N2 1 N2 2 N2 3 N2 4 N2 5 N2 6 N2 7 N2 8 pnp-1 1 pnp-1 2 pnp-1 3 pnp-1 4 pnp-1 5 pnp-1 6 pnp-1 7 pnp-1 8 Factors Sample source:Worms | Sample_class:adah-1(RNAi) Sample source:Worms | Sample_class:adah-1(RNAi) Sample source:Worms | Sample_class:adah-1(RNAi) Sample source:Worms | Sample_class:adah-1(RNAi) Sample source:Worms | Sample_class:adah-1(RNAi) Sample source:Worms | Sample_class:adah-1(RNAi) Sample source:Worms | Sample_class:adah-1(RNAi) Sample source:Worms | Sample_class:adah-1(RNAi) Sample source:Worms | Sample_class:L4440 RNAi Sample source:Worms | Sample_class:L4440 RNAi Sample source:Worms | Sample_class:L4440 RNAi Sample source:Worms | Sample_class:L4440 RNAi Sample source:Worms | Sample_class:L4440 RNAi Sample source:Worms | Sample_class:L4440 RNAi Sample source:Worms | Sample_class:L4440 RNAi Sample source:Worms | Sample_class:L4440 RNAi Sample source:Worms | Sample_class:N2 Sample source:Worms | Sample_class:N2 Sample source:Worms | Sample_class:N2 Sample source:Worms | Sample_class:N2 Sample source:Worms | Sample_class:N2 Sample source:Worms | Sample_class:N2 Sample source:Worms | Sample_class:N2 Sample source:Worms | Sample_class:N2 Sample source:Worms | Sample_class:pnp-1(jy90) Sample source:Worms | Sample_class:pnp-1(jy90) Sample source:Worms | Sample_class:pnp-1(jy90) Sample source:Worms | Sample_class:pnp-1(jy90) Sample source:Worms | Sample_class:pnp-1(jy90) Sample source:Worms | Sample_class:pnp-1(jy90) Sample source:Worms | Sample_class:pnp-1(jy90) Sample source:Worms | Sample_class:pnp-1(jy90) adenosine 8427012.1850 22583844.4300 13521453.4700 17860463.0400 10368093.3500 17355091.5100 84252802.5900 66929147.8200 3292456.2160 1812573.9040 2686590.6830 4465706.3470 1992716.3350 4674394.1900 1897.7660 5121545.1800 3796934.7880 868738.2373 2425328.3990 3910553.8060 2685210.4620 3699732.8220 3434990.1610 2991174.9090 5312897.2210 4908255.8200 5255742.8580 5016432.3610 5653922.4420 4055204.7600 5024139.8620 5133258.3880 hypoxanthine 102604887.1000 116960112.9000 72888852.5900 155255269.4000 78855270.9300 172264507.5000 102514149.7000 140764938.5000 549982747.6000 298144414.3000 279840355.5000 859877781.9000 312173413.4000 545808658.5000 1112053437.0000 404970677.1000 2109710121.0000 693242920.8000 1671157818.0000 1777502283.0000 2206088330.0000 2120682602.0000 2145740804.0000 1929689953.0000 146891050.9000 103004082.4000 77459757.0500 106792734.6000 116677100.7000 110305464.3000 157153678.9000 120190400.0000 inosine 61907113.1100 98501775.6300 76881215.5900 144370536.8000 86149695.6300 129017272.8000 208104174.6000 247199713.5000 596891230.7000 296323201.8000 296899687.2000 1571297138.0000 286213331.2000 758660770.4000 2170466423.0000 575448611.6000 1322978320.0000 390980068.4000 767421642.8000 1392862731.0000 953658215.0000 898274830.0000 1052908711.0000 619478516.9000 3714069833.0000 3591099944.0000 3057329085.0000 3583113268.0000 4461295778.0000 2716414672.0000 3688345204.0000 3367379961.0000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant adenosine 60961 C00212 7.385 266.08 hypoxanthine 135398638 C00262 2.817 135.03 inosine C00294 6.175 267.07 METABOLITES_END #END