#METABOLOMICS WORKBENCH j.a.gil.quintero@rug.nl_20160907_023019 DATATRACK_ID:719 VERSION 1 CREATED_ON November 17, 2016, 3:22 pm #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 #STUDY ST:STUDY_TITLE Exahustive degradation of nucleotide triphosphates ST:STUDY_TYPE Comparison of degradation kinetics ST:STUDY_SUMMARY The degradation kinetics of nucleotide triphosphates (ATP, GTP, UTP and CTP) ST:STUDY_SUMMARY were evaluated under boiling ethanol extraction conditions (95°C) during 0 to ST:STUDY_SUMMARY 300 minutes. 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 #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 - AG070714_3 Time (mins):- | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_4 Time (mins):- | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_5 Time (mins):- | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_6 Time (mins):- | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_7 Time (mins):- | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_8 Time (mins):- | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_10 Time (mins):5 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_11 Time (mins):5 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_12 Time (mins):5 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_13 Time (mins):5 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_14 Time (mins):5 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_15 Time (mins):5 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_17 Time (mins):10 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_18 Time (mins):10 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_19 Time (mins):10 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_20 Time (mins):10 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_21 Time (mins):10 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_22 Time (mins):10 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_24 Time (mins):20 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_25 Time (mins):20 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_26 Time (mins):20 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_27 Time (mins):20 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_28 Time (mins):20 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_29 Time (mins):20 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_31 Time (mins):30 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_32 Time (mins):30 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_33 Time (mins):30 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_34 Time (mins):30 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_35 Time (mins):30 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_36 Time (mins):30 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_39 Time (mins):60 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_40 Time (mins):60 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_41 Time (mins):60 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_42 Time (mins):60 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_43 Time (mins):60 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_44 Time (mins):60 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_46 Time (mins):120 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_47 Time (mins):120 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_48 Time (mins):120 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_49 Time (mins):120 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_50 Time (mins):120 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_51 Time (mins):120 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_53 Time (mins):180 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_54 Time (mins):180 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_55 Time (mins):180 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_56 Time (mins):180 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_57 Time (mins):180 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_58 Time (mins):180 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_60 Time (mins):240 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_61 Time (mins):240 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_62 Time (mins):240 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_63 Time (mins):240 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_64 Time (mins):240 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_65 Time (mins):240 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_67 Time (mins):300 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_68 Time (mins):300 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_69 Time (mins):300 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_70 Time (mins):300 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_71 Time (mins):300 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG070714_72 Time (mins):300 | Nucleotide:ATP SUBJECT_SAMPLE_FACTORS - AG220714_3 Time (mins):- | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_5 Time (mins):- | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_6 Time (mins):- | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_8 Time (mins):- | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_10 Time (mins):5 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_12 Time (mins):5 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_13 Time (mins):5 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_15 Time (mins):5 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_17 Time (mins):10 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_19 Time (mins):10 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_20 Time (mins):10 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_22 Time (mins):10 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_24 Time (mins):20 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_26 Time (mins):20 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_27 Time (mins):20 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_29 Time (mins):20 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_31 Time (mins):30 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_33 Time (mins):30 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_34 Time (mins):30 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_36 Time (mins):30 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_39 Time (mins):60 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_41 Time (mins):60 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_42 Time (mins):60 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_44 Time (mins):60 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_46 Time (mins):120 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_48 Time (mins):120 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_49 Time (mins):120 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_51 Time (mins):120 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_53 Time (mins):180 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_55 Time (mins):180 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_56 Time (mins):180 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_58 Time (mins):180 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_60 Time (mins):240 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_62 Time (mins):240 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_63 Time (mins):240 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_65 Time (mins):240 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_67 Time (mins):300 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_69 Time (mins):300 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_70 Time (mins):300 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG220714_72 Time (mins):300 | Nucleotide:GTP SUBJECT_SAMPLE_FACTORS - AG240714_3 Time (mins):- | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_4 Time (mins):- | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_5 Time (mins):- | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_6 Time (mins):- | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_8 Time (mins):5 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_9 Time (mins):5 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_10 Time (mins):5 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_11 Time (mins):5 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_13 Time (mins):10 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_14 Time (mins):10 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_15 Time (mins):10 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_16 Time (mins):10 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_18 Time (mins):20 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_19 Time (mins):20 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_20 Time (mins):20 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_21 Time (mins):20 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_23 Time (mins):30 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_24 Time (mins):30 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_25 Time (mins):30 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_26 Time (mins):30 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_29 Time (mins):60 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_30 Time (mins):60 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_31 Time (mins):60 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_32 Time (mins):60 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_34 Time (mins):120 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_35 Time (mins):120 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_36 Time (mins):120 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_37 Time (mins):120 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_39 Time (mins):180 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_40 Time (mins):180 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_41 Time (mins):180 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_42 Time (mins):180 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_44 Time (mins):240 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_45 Time (mins):240 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_46 Time (mins):240 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_47 Time (mins):240 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_49 Time (mins):300 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_50 Time (mins):300 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_51 Time (mins):300 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG240714_52 Time (mins):300 | Nucleotide:UTP SUBJECT_SAMPLE_FACTORS - AG260714_3 Time (mins):- | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_4 Time (mins):- | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_5 Time (mins):- | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_6 Time (mins):- | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_7 Time (mins):5 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_8 Time (mins):5 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_9 Time (mins):5 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_10 Time (mins):5 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_11 Time (mins):10 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_12 Time (mins):10 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_13 Time (mins):10 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_14 Time (mins):10 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_15 Time (mins):20 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_16 Time (mins):20 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_17 Time (mins):20 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_18 Time (mins):20 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_19 Time (mins):30 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_20 Time (mins):30 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_21 Time (mins):30 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_22 Time (mins):30 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_25 Time (mins):60 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_26 Time (mins):60 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_27 Time (mins):60 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_28 Time (mins):60 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_29 Time (mins):120 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_30 Time (mins):120 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_31 Time (mins):120 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_32 Time (mins):120 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_33 Time (mins):180 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_34 Time (mins):180 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_35 Time (mins):180 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_36 Time (mins):180 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_37 Time (mins):240 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_38 Time (mins):240 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_39 Time (mins):240 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_40 Time (mins):240 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_41 Time (mins):300 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_42 Time (mins):300 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_43 Time (mins):300 | Nucleotide:CTP SUBJECT_SAMPLE_FACTORS - AG260714_44 Time (mins):300 | Nucleotide:CTP #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:CHROMATOGRAPHY_TYPE HILIC MS:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Phenomenex Luna NH2 column (3 µm, 100 × 2 mm) 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:COLUMN_TEMPERATURE 20C CH:SOLVENT_A 5 mM ammonium acetate in water at pH 9.9 CH:SOLVENT_B Acetonitrile CH:SAMPLE_INJECTION 10 µL CH:ANALYTICAL_TIME 20 min #ANALYSIS AN:ANALYSIS_TYPE MS AN:SOFTWARE_VERSION MassLynx 4.1 #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt-G2-Si 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 AG070714_3 AG070714_4 AG070714_5 AG070714_6 AG070714_7 AG070714_8 AG070714_10 AG070714_11 AG070714_12 AG070714_13 AG070714_14 AG070714_15 AG070714_17 AG070714_18 AG070714_19 AG070714_20 AG070714_21 AG070714_22 AG070714_24 AG070714_25 AG070714_26 AG070714_27 AG070714_28 AG070714_29 AG070714_31 AG070714_32 AG070714_33 AG070714_34 AG070714_35 AG070714_36 AG070714_39 AG070714_40 AG070714_41 AG070714_42 AG070714_43 AG070714_44 AG070714_46 AG070714_47 AG070714_48 AG070714_49 AG070714_50 AG070714_51 AG070714_53 AG070714_54 AG070714_55 AG070714_56 AG070714_57 AG070714_58 AG070714_60 AG070714_61 AG070714_62 AG070714_63 AG070714_64 AG070714_65 AG070714_67 AG070714_68 AG070714_69 AG070714_70 AG070714_71 AG070714_72 AG220714_3 AG220714_5 AG220714_6 AG220714_8 AG220714_10 AG220714_12 AG220714_13 AG220714_15 AG220714_17 AG220714_19 AG220714_20 AG220714_22 AG220714_24 AG220714_26 AG220714_27 AG220714_29 AG220714_31 AG220714_33 AG220714_34 AG220714_36 AG220714_39 AG220714_41 AG220714_42 AG220714_44 AG220714_46 AG220714_48 AG220714_49 AG220714_51 AG220714_53 AG220714_55 AG220714_56 AG220714_58 AG220714_60 AG220714_62 AG220714_63 AG220714_65 AG220714_67 AG220714_69 AG220714_70 AG220714_72 AG240714_3 AG240714_4 AG240714_5 AG240714_6 AG240714_8 AG240714_9 AG240714_10 AG240714_11 AG240714_13 AG240714_14 AG240714_15 AG240714_16 AG240714_18 AG240714_19 AG240714_20 AG240714_21 AG240714_23 AG240714_24 AG240714_25 AG240714_26 AG240714_29 AG240714_30 AG240714_31 AG240714_32 AG240714_34 AG240714_35 AG240714_36 AG240714_37 AG240714_39 AG240714_40 AG240714_41 AG240714_42 AG240714_44 AG240714_45 AG240714_46 AG240714_47 AG240714_49 AG240714_50 AG240714_51 AG240714_52 AG260714_3 AG260714_4 AG260714_5 AG260714_6 AG260714_7 AG260714_8 AG260714_9 AG260714_10 AG260714_11 AG260714_12 AG260714_13 AG260714_14 AG260714_15 AG260714_16 AG260714_17 AG260714_18 AG260714_19 AG260714_20 AG260714_21 AG260714_22 AG260714_25 AG260714_26 AG260714_27 AG260714_28 AG260714_29 AG260714_30 AG260714_31 AG260714_32 AG260714_33 AG260714_34 AG260714_35 AG260714_36 AG260714_37 AG260714_38 AG260714_39 AG260714_40 AG260714_41 AG260714_42 AG260714_43 AG260714_44 Factors Time (mins):- | Nucleotide:ATP Time (mins):- | Nucleotide:ATP Time (mins):- | Nucleotide:ATP Time (mins):- | Nucleotide:ATP Time (mins):- | Nucleotide:ATP Time (mins):- | Nucleotide:ATP Time (mins):5 | Nucleotide:ATP Time (mins):5 | Nucleotide:ATP Time (mins):5 | Nucleotide:ATP Time (mins):5 | Nucleotide:ATP Time (mins):5 | Nucleotide:ATP Time (mins):5 | Nucleotide:ATP Time (mins):10 | Nucleotide:ATP Time (mins):10 | Nucleotide:ATP Time (mins):10 | Nucleotide:ATP Time (mins):10 | Nucleotide:ATP Time (mins):10 | Nucleotide:ATP Time (mins):10 | Nucleotide:ATP Time (mins):20 | Nucleotide:ATP Time (mins):20 | Nucleotide:ATP Time (mins):20 | Nucleotide:ATP Time (mins):20 | Nucleotide:ATP Time (mins):20 | Nucleotide:ATP Time (mins):20 | Nucleotide:ATP Time (mins):30 | Nucleotide:ATP Time (mins):30 | Nucleotide:ATP Time (mins):30 | Nucleotide:ATP Time (mins):30 | Nucleotide:ATP Time (mins):30 | Nucleotide:ATP Time (mins):30 | Nucleotide:ATP Time (mins):60 | Nucleotide:ATP Time (mins):60 | Nucleotide:ATP Time (mins):60 | Nucleotide:ATP Time (mins):60 | Nucleotide:ATP Time (mins):60 | Nucleotide:ATP Time (mins):60 | Nucleotide:ATP Time (mins):120 | Nucleotide:ATP Time (mins):120 | Nucleotide:ATP Time (mins):120 | Nucleotide:ATP Time (mins):120 | Nucleotide:ATP Time (mins):120 | Nucleotide:ATP Time (mins):120 | Nucleotide:ATP Time (mins):180 | Nucleotide:ATP Time (mins):180 | Nucleotide:ATP Time (mins):180 | Nucleotide:ATP Time (mins):180 | Nucleotide:ATP Time (mins):180 | Nucleotide:ATP Time (mins):180 | Nucleotide:ATP Time (mins):240 | Nucleotide:ATP Time (mins):240 | Nucleotide:ATP Time (mins):240 | Nucleotide:ATP Time (mins):240 | Nucleotide:ATP Time (mins):240 | Nucleotide:ATP Time (mins):240 | Nucleotide:ATP Time (mins):300 | Nucleotide:ATP Time (mins):300 | Nucleotide:ATP Time (mins):300 | Nucleotide:ATP Time (mins):300 | Nucleotide:ATP Time (mins):300 | Nucleotide:ATP Time (mins):300 | Nucleotide:ATP Time (mins):- | Nucleotide:GTP Time (mins):- | Nucleotide:GTP Time (mins):- | Nucleotide:GTP Time (mins):- | Nucleotide:GTP Time (mins):5 | Nucleotide:GTP Time (mins):5 | Nucleotide:GTP Time (mins):5 | Nucleotide:GTP Time (mins):5 | Nucleotide:GTP Time (mins):10 | Nucleotide:GTP Time (mins):10 | Nucleotide:GTP Time (mins):10 | Nucleotide:GTP Time (mins):10 | Nucleotide:GTP Time (mins):20 | Nucleotide:GTP Time (mins):20 | Nucleotide:GTP Time (mins):20 | Nucleotide:GTP Time (mins):20 | Nucleotide:GTP Time (mins):30 | Nucleotide:GTP Time (mins):30 | Nucleotide:GTP Time (mins):30 | Nucleotide:GTP Time (mins):30 | Nucleotide:GTP Time (mins):60 | Nucleotide:GTP Time (mins):60 | Nucleotide:GTP Time (mins):60 | Nucleotide:GTP Time (mins):60 | Nucleotide:GTP Time (mins):120 | Nucleotide:GTP Time (mins):120 | Nucleotide:GTP Time (mins):120 | Nucleotide:GTP Time (mins):120 | Nucleotide:GTP Time (mins):180 | Nucleotide:GTP Time (mins):180 | Nucleotide:GTP Time (mins):180 | Nucleotide:GTP Time (mins):180 | Nucleotide:GTP Time (mins):240 | Nucleotide:GTP Time (mins):240 | Nucleotide:GTP Time (mins):240 | Nucleotide:GTP Time (mins):240 | Nucleotide:GTP Time (mins):300 | Nucleotide:GTP Time (mins):300 | Nucleotide:GTP Time (mins):300 | Nucleotide:GTP Time (mins):300 | Nucleotide:GTP Time (mins):- | Nucleotide:UTP Time (mins):- | Nucleotide:UTP Time (mins):- | Nucleotide:UTP Time (mins):- | Nucleotide:UTP Time (mins):5 | Nucleotide:UTP Time (mins):5 | Nucleotide:UTP Time (mins):5 | Nucleotide:UTP Time (mins):5 | Nucleotide:UTP Time (mins):10 | Nucleotide:UTP Time (mins):10 | Nucleotide:UTP Time (mins):10 | Nucleotide:UTP Time (mins):10 | Nucleotide:UTP Time (mins):20 | Nucleotide:UTP Time (mins):20 | Nucleotide:UTP Time (mins):20 | Nucleotide:UTP Time (mins):20 | Nucleotide:UTP Time (mins):30 | Nucleotide:UTP Time (mins):30 | Nucleotide:UTP Time (mins):30 | Nucleotide:UTP Time (mins):30 | Nucleotide:UTP Time (mins):60 | Nucleotide:UTP Time (mins):60 | Nucleotide:UTP Time (mins):60 | Nucleotide:UTP Time (mins):60 | Nucleotide:UTP Time (mins):120 | Nucleotide:UTP Time (mins):120 | Nucleotide:UTP Time (mins):120 | Nucleotide:UTP Time (mins):120 | Nucleotide:UTP Time (mins):180 | Nucleotide:UTP Time (mins):180 | Nucleotide:UTP Time (mins):180 | Nucleotide:UTP Time (mins):180 | Nucleotide:UTP Time (mins):240 | Nucleotide:UTP Time (mins):240 | Nucleotide:UTP Time (mins):240 | Nucleotide:UTP Time (mins):240 | Nucleotide:UTP Time (mins):300 | Nucleotide:UTP Time (mins):300 | Nucleotide:UTP Time (mins):300 | Nucleotide:UTP Time (mins):300 | Nucleotide:UTP Time (mins):- | Nucleotide:CTP Time (mins):- | Nucleotide:CTP Time (mins):- | Nucleotide:CTP Time (mins):- | Nucleotide:CTP Time (mins):5 | Nucleotide:CTP Time (mins):5 | Nucleotide:CTP Time (mins):5 | Nucleotide:CTP Time (mins):5 | Nucleotide:CTP Time (mins):10 | Nucleotide:CTP Time (mins):10 | Nucleotide:CTP Time (mins):10 | Nucleotide:CTP Time (mins):10 | Nucleotide:CTP Time (mins):20 | Nucleotide:CTP Time (mins):20 | Nucleotide:CTP Time (mins):20 | Nucleotide:CTP Time (mins):20 | Nucleotide:CTP Time (mins):30 | Nucleotide:CTP Time (mins):30 | Nucleotide:CTP Time (mins):30 | Nucleotide:CTP Time (mins):30 | Nucleotide:CTP Time (mins):60 | Nucleotide:CTP Time (mins):60 | Nucleotide:CTP Time (mins):60 | Nucleotide:CTP Time (mins):60 | Nucleotide:CTP Time (mins):120 | Nucleotide:CTP Time (mins):120 | Nucleotide:CTP Time (mins):120 | Nucleotide:CTP Time (mins):120 | Nucleotide:CTP Time (mins):180 | Nucleotide:CTP Time (mins):180 | Nucleotide:CTP Time (mins):180 | Nucleotide:CTP Time (mins):180 | Nucleotide:CTP Time (mins):240 | Nucleotide:CTP Time (mins):240 | Nucleotide:CTP Time (mins):240 | Nucleotide:CTP Time (mins):240 | Nucleotide:CTP Time (mins):300 | Nucleotide:CTP Time (mins):300 | Nucleotide:CTP Time (mins):300 | Nucleotide:CTP Time (mins):300 | Nucleotide:CTP ATP 151858.2 84425.75 78593.72 50148.59 114408.7 76767.35 114510.5 48828.38 178695.7 129567.6 231155.2 125460.3 102001.1 74218.66 243411.7 157613.8 134816.7 78633.19 222030.2 156345.5 149268.5 104856.8 137182.6 110160.2 177238.2 121920.3 79861.75 59770.29 108841.5 77178.27 68602.84 52276.95 67136.62 56672.37 68591.83 54075.15 37075.96 29757.29 10386.06 8349.233 54830.51 35623 614.028 446.234 304.383 296.582 337.008 256.984 187.912 187.215 118.194 70.254 57.474 185.906 102.867 88.714 193.276 181.706 144.898 85.945 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N ADP 8444.877 5008.128 4663.816 3047.472 5933.581 4495.27 19097.42 9078.881 23923.27 17110.4 31094.58 17461.72 23235.56 16805.33 50267.32 32420.32 30499.16 20269.11 72248.13 51587.84 40282.68 30500.47 40883.01 34857.63 85599.14 62762.96 37055.87 29506.02 51566.15 40102.61 53392.11 39886.84 53658.89 46853.35 51969.81 41773.88 66920.52 54938.04 51771.18 46075.2 88790.41 59199.98 9630.116 8692.435 7129.956 6569.185 9285.517 8318.671 3956.37 3025.973 2642.759 2255.563 3004.277 2518.905 3160.105 2459.474 2246.495 1772.998 2049.655 1805.984 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N AMP 469.829 295.336 396.543 243.521 398.189 300.143 986.537 524.514 1370.299 1127.366 1693.498 1065.75 1574.032 1272.026 2972.262 1912.357 1670.703 1400.063 6105.836 4333.425 3335.85 2931.157 3975.908 3384.687 10847.47 7879.956 4602.556 3846.474 5836.747 5041.39 9971.78 7741.357 10732.41 9385.273 8982.024 8032.475 25640.5 21907.7 51455.89 44503.57 35003.39 25024.88 103756.3 88317.43 87364.05 77005.76 91438.53 79985.45 92499.67 80440.97 72951.61 67583.22 83651.25 75503.32 91572.78 77649.51 81865.75 71678.62 77480.7 72664.35 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N GTP N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N 37380.58 49636.21 43227.8 43035.93 44631.43 45481.54 39193.36 44460.94 41542.54 44595.51 39166.99 40413.74 42375.55 42177.31 42212.43 44217.77 39071.18 41412.24 50114.44 59251.3 37809.16 40161.61 36441.2 36401.49 19694.98 4538.063 3548.285 25972.08 1463.03 967.748 769.109 1101.448 439.48 296.5 330.72 264.612 204.153 145.164 101.492 88.879 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N GDP N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N 4338.394 4923.839 4337.756 4154.628 6592.702 5822.201 5262.38 5821.242 6864.353 6401.574 6363.355 9605.273 11856.1 9158.078 9253.56 8598.909 9366.334 10873.37 13667.91 14286.32 14734.76 17269.14 15240.5 16000.57 32658.53 27682.64 26152.87 29489.01 12064.57 6805.84 6710.771 14173.82 3135.222 3060.341 2306.646 1512.823 832.618 672.009 632.474 817.247 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N GMP N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N 248.722 238.098 213.374 150.728 281.32 279.018 307.569 330.202 404.572 430.278 372.532 1621.674 1210.596 2947.213 2971.204 850.941 1617.103 829.181 1040.469 1171.878 3375.267 2312.359 2235.733 2256.375 11774.32 32157.4 29114.69 8887.814 50713.02 51734.02 51189.66 52865.23 59625.87 61817.55 53906.45 50237.46 50611.32 46717.7 44602.92 47600.81 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N UTP N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N 68479.94 23168.93 23430.03 26418.79 26991.49 26345.52 28008.42 28000.34 24855.33 27211.64 27611.4 28520.11 27375.29 27479.99 24698.66 26165.03 26383.31 27495.17 25575.34 31951 25374.37 26353.9 21809.31 26562.83 19035.23 20015.18 15033.57 20272.58 12068.92 11107.73 11806.05 11887.28 7493.07 7040.287 6319.574 7246.219 3524.003 3799.301 2741.851 3606.811 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N UDP N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N 10366.96 3662.992 3594.252 4173.576 5530.742 5789.907 5778.873 5193.706 6386.311 7817.971 7302.802 7417.116 8900.235 8616.185 7703.434 8305.348 10110 11111.92 10706.19 12319.36 16040.63 16252.52 15086.55 16825.17 24735.68 24881.59 21635.13 25151.54 26869.84 26254.81 24044.18 27161.84 29845.82 28268.14 26044.91 28506.2 26715.77 26286.46 20780.57 24031.53 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N UMP N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N 413.986 166.563 171.557 74.338 271.198 265.713 204.792 229.146 334.708 533.454 412.426 419.967 653.697 729.582 651.517 703.921 1157.23 1121.318 1103.522 1187.038 2543.866 2955.88 2532.504 3033.36 9117.534 9581.531 8022.63 9462.476 16071.5 15885.6 15970.04 16302.97 26506.07 24779.33 23271.83 25903.58 41626.32 37353.08 31115.5 35122.45 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N CTP N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N 43936.39 49308.77 42537.46 47137.42 47711.78 43559.17 36460.44 49297.61 50102.54 47763.18 40124.36 56599.31 32204.17 36701.34 36179.83 50485.95 54916.44 34157.99 36032.96 40548.39 34311.87 34944.66 32054.1 35084.91 21385.58 21411.23 19425.23 11644.69 16493.61 11540.99 11494.01 11995.36 8654.965 8657.412 6805.326 7367.804 2670.041 2195.195 2079.084 4805.602 CDP N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N 4149.242 4143.203 4186.939 4533.104 7026.141 6937.559 5606.611 7171.375 10011.63 9966.227 8164.041 11355.83 7694.54 10227.78 10731.36 14599.16 17608.72 12393.13 10953.97 13874.9 22586.73 21968.36 19291.26 20556.61 31519.27 31413.33 28763.49 31386.89 35567.43 31585.77 30511.79 29932.29 30376.42 32898.74 26842.82 28502.14 22970.41 20868.8 20365.84 25381.73 CMP N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N 248.602 271.852 265.284 226.371 457.845 446.076 342.407 438.013 705.373 641.211 541.73 704.073 685.405 777.593 773.279 1049.468 1556.685 1095.728 1052.66 1234.495 3364.436 3322.017 2995.752 3094.432 8973.604 10687.06 10142.96 13097.24 16630.21 17924.19 15796.76 17223.71 22817.99 25523.35 21944.67 23260.39 30337.02 32757.42 31959.76 29309.21 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID ATP 505.988 5957 C00002 ADP 426.022 6022 C00008 AMP 346.055 6083 C00020 GTP 521.983 6830 C00044 GDP 442.017 8977 C00035 GMP 362.051 6804 C00144 UTP 482.961 6133 C00075 UDP 402.995 6031 C00015 UMP 323.029 6030 C00105 CTP 481.977 6176 C00063 CDP 402.011 6132 C00112 CMP 322.045 6131 C00055 METABOLITES_END #END