#METABOLOMICS WORKBENCH kenanazam_20160913_123734 DATATRACK_ID:740 STUDY_ID:ST000472 ANALYSIS_ID:AN001823 VERSION 1 CREATED_ON 08-09-2023 #PROJECT PR:PROJECT_TITLE Degradation of Nucleotides under different chemical environments PR:PROJECT_SUMMARY The degradation kinetics of nucleotide triphosphates (ATP, GTP, UTP and CTP) PR:PROJECT_SUMMARY were evaluated under boiling ethanol extraction conditions (95°C) during 0 to PR:PROJECT_SUMMARY 300 minutes. PR:INSTITUTE University of Groningen PR:DEPARTMENT Analytical Biochemistry PR:LAST_NAME Bischoff PR:FIRST_NAME Rainer PR:ADDRESS Antonius Deusinglaan 1 (XB20), Building 3226, room 601, 9713 AV Groningen, The PR:ADDRESS Netherlands PR:EMAIL r.p.h.bischoff@rug.nl PR:PHONE NA PR:PUBLICATIONS Gil, A., Siegel, D., Bonsing-Vedelaar, S. et al. The degradation of nucleotide PR:PUBLICATIONS triphosphates extracted under boiling ethanol conditions is prevented by the PR:PUBLICATIONS yeast cellular matrix. Metabolomics (2017) 13:1. doi:10.1007/s11306-016-1140-4 PR:DOI http://dx.doi.org/10.21228/M8CW21 #STUDY ST:STUDY_TITLE Effect of the culture medium on the degradation of nucleotide triphosphates ST:STUDY_TYPE Endpoint measurement ST:STUDY_SUMMARY The effect of the Verduyn culture medium on the degradation profiles of ST:STUDY_SUMMARY nucleotide triphosphates extracted under typical boiling ethanol conditions was ST:STUDY_SUMMARY evaluated. ST:INSTITUTE University of Groningen ST:DEPARTMENT Analytical Biochemistry ST:LAST_NAME Bischoff ST:FIRST_NAME Rainer ST:ADDRESS Antonius Deusinglaan 1 (XB20), Building 3226, room 601, 9713 AV Groningen, The ST:ADDRESS Netherlands ST:EMAIL r.p.h.bischoff@rug.nl ST:PHONE NA ST:SUBMIT_DATE 2016-09-13 #SUBJECT SU:SUBJECT_TYPE Chemical SU:SUBJECT_SPECIES None SU:SUBJECT_COMMENTS Primary standards of nucleotide triphosphates (ATP, GTP, UTP and CTP). #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - AG250615_23 Time (mins):15 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG250615_24 Time (mins):15 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG250615_25 Time (mins):15 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG250615_26 Time (mins):15 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG150715_66 Time (mins):15 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG150715_68 Time (mins):15 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG150715_70 Time (mins):15 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG150715_72 Time (mins):15 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG150715_37 Time (mins):15 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG150715_38 Time (mins):15 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG150715_39 Time (mins):15 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG150715_40 Time (mins):15 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG200715_15 Time (mins):15 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG200715_17 Time (mins):15 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG200715_19 Time (mins):15 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG200715_21 Time (mins):15 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG250615_18 Time (mins):- | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG250615_19 Time (mins):- | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG250615_20 Time (mins):- | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG250615_21 Time (mins):- | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG150715_58 Time (mins):- | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG150715_60 Time (mins):- | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG150715_62 Time (mins):- | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG150715_64 Time (mins):- | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG150715_32 Time (mins):- | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG150715_33 Time (mins):- | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG150715_34 Time (mins):- | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG150715_35 Time (mins):- | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG200715_11 Time (mins):- | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG200715_13 Time (mins):- | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG200715_7 Time (mins):- | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG200715_9 Time (mins):- | Nucleotide:UTP #COLLECTION CO:COLLECTION_SUMMARY None #TREATMENT TR:TREATMENT_SUMMARY Pure solutions of nucleotide triphosphates (ATP, GTP, CTP and UTP) were TR:TREATMENT_SUMMARY incubated under boiling ethanol conditions (95°C) during 0, 5, 10, 20, 30, 60, TR:TREATMENT_SUMMARY 120, 180, 240 and 300 minutes. TR:TREATMENT Abiotic #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Tubes containing 496 µL of 75% (aq) ethanol were preheated at 95°C for 5 min, SP:SAMPLEPREP_SUMMARY followed by the addition of 4 µL of each nucleotide standard solution (500 µM) SP:SAMPLEPREP_SUMMARY and vigorous mixing. 4 µL of solutions “A” to “H” were added to the SP:SAMPLEPREP_SUMMARY reaction mixture and the samples were incubated at 95°C under shaking for 0 and SP:SAMPLEPREP_SUMMARY 15 min. Reactions were stopped by snap-freezing in liquid nitrogen and samples SP:SAMPLEPREP_SUMMARY were stored on dry ice. Subsequently, samples were thawed and 20 µL of the SP:SAMPLEPREP_SUMMARY 13C15N-labeled internal standard solution were added for quantitative analysis. SP:SAMPLEPREP_SUMMARY Excess solvent was evaporated under a stream of nitrogen without heating. SP:SAMPLEPREP_SUMMARY Finally, samples were reconstituted in 200 µL of acetonitrile-water 70:30 and SP:SAMPLEPREP_SUMMARY stored at –40°C until analysis by LC-MS. After the experimental procedure the SP:SAMPLEPREP_SUMMARY final concentration of all components in solutions “A” to “H” was 10 SP:SAMPLEPREP_SUMMARY µM. Compounds to be tested were divided into eight groups containing SP:SAMPLEPREP_SUMMARY approximately ten compounds each, mainly comprising major central carbon SP:SAMPLEPREP_SUMMARY metabolites including amino acids, organic acids, sugar phosphates, coenzymes, SP:SAMPLEPREP_SUMMARY etc (Table 1). SP:PROCESSING_METHOD Preparation of reagents, dilution, solvent removal under a stream of nitrogen SP:PROCESSING_METHOD without heating and reconstitution SP:EXTRACTION_METHOD Boiling ethanol (95°C) SP:EXTRACT_ENRICHMENT Evaporation of excess of solvent under a stream of nitrogen without heating. SP:EXTRACT_STORAGE -40C SP:SAMPLE_RESUSPENSION 200 µL of acetonitrile-water 70:30 #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Untargeted HILIC Method CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Phenomenex Luna NH2 (100 x 2.0mm,3um) CH:COLUMN_TEMPERATURE 20C CH:FLOW_GRADIENT 30% eluent A to 99% eluent A in 8 min, followed by isocratic elution at 99% CH:FLOW_GRADIENT eluent A until 14 min. A conditioning cycle of 6 min with the initial CH:FLOW_GRADIENT proportions of eluents A and B was performed prior to the next analysis. CH:FLOW_RATE 0.25 mL/min CH:SAMPLE_INJECTION 10 µL CH:SOLVENT_A 100% water; 5 mM ammonium acetate, pH 9.9 CH:SOLVENT_B 100% acetonitrile CH:ANALYTICAL_TIME 20 min CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS AN:SOFTWARE_VERSION MassLynx 4.1 #MS MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:CAPILLARY_VOLTAGE 2KV MS:COLLISION_ENERGY 2 V for the low-collision energy scan, and 10–30 V for the high-collision MS:COLLISION_ENERGY energy scan MS:DRY_GAS_FLOW Argon MS:SOURCE_TEMPERATURE 150C MS:DESOLVATION_TEMPERATURE 400C MS:SCAN_RANGE_MOVERZ 50 to 1200 Da #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples AG250615_23 AG250615_24 AG250615_25 AG250615_26 AG150715_66 AG150715_68 AG150715_70 AG150715_72 AG150715_37 AG150715_38 AG150715_39 AG150715_40 AG200715_15 AG200715_17 AG200715_19 AG200715_21 AG250615_18 AG250615_19 AG250615_20 AG250615_21 AG150715_58 AG150715_60 AG150715_62 AG150715_64 AG150715_32 AG150715_33 AG150715_34 AG150715_35 AG200715_11 AG200715_13 AG200715_7 AG200715_9 Factors Time (mins):15 | Nucleotide:ATP Time (mins):15 | Nucleotide:ATP Time (mins):15 | Nucleotide:ATP Time (mins):15 | Nucleotide:ATP Time (mins):15 | Nucleotide:CTP Time (mins):15 | Nucleotide:CTP Time (mins):15 | Nucleotide:CTP Time (mins):15 | Nucleotide:CTP Time (mins):15 | Nucleotide:GTP Time (mins):15 | Nucleotide:GTP Time (mins):15 | Nucleotide:GTP Time (mins):15 | Nucleotide:GTP Time (mins):15 | Nucleotide:UTP Time (mins):15 | Nucleotide:UTP Time (mins):15 | Nucleotide:UTP Time (mins):15 | Nucleotide:UTP Time (mins):- | Nucleotide:ATP Time (mins):- | Nucleotide:ATP Time (mins):- | Nucleotide:ATP Time (mins):- | Nucleotide:ATP Time (mins):- | Nucleotide:CTP Time (mins):- | Nucleotide:CTP Time (mins):- | Nucleotide:CTP Time (mins):- | Nucleotide:CTP Time (mins):- | Nucleotide:GTP Time (mins):- | Nucleotide:GTP Time (mins):- | Nucleotide:GTP Time (mins):- | Nucleotide:GTP Time (mins):- | Nucleotide:UTP Time (mins):- | Nucleotide:UTP Time (mins):- | Nucleotide:UTP Time (mins):- | Nucleotide:UTP ADP 2360.8840 12907.6100 3465.6190 1945.9350 1903.5810 927.9810 1291.4800 822.7040 ADP 13C15N IS 43450.0500 274557.1000 59315.5900 29175.0400 58526.8800 28708.8800 37737.8800 28798.2900 AMP 177.5120 663.1760 502.0780 278.4270 273.8330 231.3310 157.5570 247.4240 AMP 13C15N IS 5641.5440 19936.3000 9889.3900 5298.6910 7883.9380 4390.7530 5677.5900 4816.0640 ATP 8946.1260 115064.5000 12721.4900 7043.7920 15872.6700 6659.9530 7629.2530 6426.9920 ATP 13C15N IS 6792.0310 95906.4800 9591.3970 4575.3110 12307.0600 5124.4480 6352.1990 4782.4500 CDP 3735.5330 893.8380 5033.5200 7496.9110 6471.4830 5317.3670 3520.0790 571.1300 CDP 13C15N IS 107048.1000 12381.5600 114564.6000 170053.9000 267330.4000 203322.7000 148313.5000 6861.8590 CMP 36.5410 21.8880 39.4010 22.7720 42.5700 24.2900 20.4530 3.9750 CMP 13C15N IS 20522.6800 12569.3600 20351.4600 25399.9700 19011.8400 32355.2700 25560.6100 9937.2470 CTP 22968.5100 3843.6170 33437.3400 50920.2500 46231.7600 45417.4800 29033.0000 2454.0090 CTP 13C15N IS 31333.0600 5615.8190 36016.4400 51916.9300 66718.6500 51889.9900 36801.0200 2623.2130 GDP 6309.9400 6816.5390 4134.6440 7710.6410 1447.6950 3696.5740 3691.8120 4539.4400 GDP 13C15N IS 43752.2300 52033.9100 25346.4900 135564.3000 14590.9400 29737.4400 39446.2500 43758.8600 GMP 164.1810 205.9250 174.4440 322.9910 87.5240 145.7360 177.9550 121.8360 GMP 13C15N IS 8796.9580 9471.1770 6562.9370 26236.9100 4212.6560 7408.2220 8245.0500 8644.0550 GTP 19927.0400 28339.2300 10777.7000 25240.8500 3064.3200 12492.4600 21716.8300 26585.8400 GTP 13C15N IS 18926.7100 27949.5300 9515.5660 59874.1600 2721.3370 9752.1430 17292.0400 24116.0600 UDP 427.4820 5806.0730 6746.2920 766.8740 710.1170 2214.2280 663.1300 674.2730 UDP 13C15N IS 7696.6680 184571.5000 187702.0000 12198.9600 19969.3500 94194.8300 12013.4800 18056.1800 UMP 17.8140 95.4060 192.2660 207.8500 31.5600 57.2210 63.8060 30.6950 UMP 13C15N IS 7520.3500 39464.4300 34373.0200 9593.7040 13451.6300 26327.3900 10817.5900 13409.3500 UTP 681.5660 15012.5900 18515.6800 890.4220 1828.2460 8144.6520 893.6130 1635.9780 UTP 13C15N IS 723.2980 17516.4900 18070.8700 941.1400 2133.9020 8850.8290 1029.1440 1883.6550 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 ADP 6022 C00008 426.022 ADP 13C15N IS AMP 6083 C00020 346.055 AMP 13C15N IS ATP 5957 C00002 505.988 ATP 13C15N IS CDP 6132 C00112 402.011 CDP 13C15N IS CMP 6131 C00055 322.045 CMP 13C15N IS CTP 6176 C00063 481.977 CTP 13C15N IS GDP 8977 C00035 442.017 GDP 13C15N IS GMP 6804 C00144 362.051 GMP 13C15N IS GTP 6830 C00044 521.983 GTP 13C15N IS UDP 6031 C00015 402.995 UDP 13C15N IS UMP 6030 C00105 323.029 UMP 13C15N IS UTP 6133 C00075 482.961 UTP 13C15N IS METABOLITES_END #END