#METABOLOMICS WORKBENCH atsai_20250205_193220 DATATRACK_ID:5596 STUDY_ID:ST003727 ANALYSIS_ID:AN006114 PROJECT_ID:PR002314 VERSION 1 CREATED_ON February 13, 2025, 5:15 pm #PROJECT PR:PROJECT_TITLE Comprehensive Nucleoside Analysis of Archaeal RNA Modification Profiles Reveals PR:PROJECT_TITLE a m7G in the Conserved P-loop of 23S rRNA PR:PROJECT_SUMMARY Extremophilic archaea employ diverse chemical RNA modifications, providing a PR:PROJECT_SUMMARY rich source of new enzymes for biotechnologically valuable RNA manipulations. PR:PROJECT_SUMMARY Our understanding of the modified nucleoside profiles in Archaea, as well as the PR:PROJECT_SUMMARY functions and dynamic regulation of specific RNA modifications is far from PR:PROJECT_SUMMARY complete. Here, we established an extensive profile of nucleoside modifications PR:PROJECT_SUMMARY in thermophilic and mesophilic Archaea through highly sensitive LC-MS/MS PR:PROJECT_SUMMARY analysis and rigorous non-coding RNA depletion, identifying - with high PR:PROJECT_SUMMARY confidence - at least four previously unannotated modifications in archaeal PR:PROJECT_SUMMARY mRNAs. Nucleoside quantification analysis conducted on total, large, small, and PR:PROJECT_SUMMARY mRNA-enriched subfractions of the model hyperthermophilic archaeon Thermococcus PR:PROJECT_SUMMARY kodakarensis revealed a series of modifications whose abundance is dynamically PR:PROJECT_SUMMARY responsive to growth temperatures, implying that specific RNA modifications are PR:PROJECT_SUMMARY fitness relevant under specific growth conditions. To predict the RNA-modifying PR:PROJECT_SUMMARY enzymes most likely to generate the new and dynamic RNA modifications, we PR:PROJECT_SUMMARY leveraged a bioinformatics analysis of open-access databases to annotate likely PR:PROJECT_SUMMARY functional domains of archaeal proteins. Putative enzyme activities were PR:PROJECT_SUMMARY confirmed in vitro and in vivo by assessing the presence of the target RNA PR:PROJECT_SUMMARY modification in genetic deletion strains of T. kodakarensis. Our approach led to PR:PROJECT_SUMMARY the discovery of a methyltransferase-encoded gene responsible for m7G PR:PROJECT_SUMMARY modification in the P-loop of 23S rRNA peptidyl transferase center and validates PR:PROJECT_SUMMARY a novel and effective platform for discovering RNA-modifying enzymes through PR:PROJECT_SUMMARY LC-MS/MS analysis that will accelerate efforts of the community towards PR:PROJECT_SUMMARY uncovering the complex and dynamic roles of RNA modifications. PR:INSTITUTE New England Biolabs PR:LAST_NAME Tsai PR:FIRST_NAME Yueh-Lin PR:ADDRESS 44 Dunham Ridge, Beverly, MA 01915 PR:EMAIL atsai@neb.com PR:PHONE 978-380-6587 #STUDY ST:STUDY_TITLE Identification of modified nucleosides in mRNA-enriched archaeal samples ST:STUDY_SUMMARY Total RNA extracted from five archaeal species were depleted with rRNAs and ST:STUDY_SUMMARY digested to nucleosides for UHPLC-QqQ analysis. ST:INSTITUTE New England Biolabs ST:LAST_NAME Tsai ST:FIRST_NAME Yueh-Lin ST:ADDRESS 44 Dunham Ridge, Beverly, MA 01915 ST:EMAIL atsai@neb.com ST:PHONE 978-380-6587 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Thermococcus kodakarensis, Thermococcus sp. AM4, Methanococcus maripaludis, SU:SUBJECT_SPECIES Sulfolobus acidocaldarius, Sulfolobus islandicus SU:TAXONOMY_ID 311400, 246969, 39152, 2285, 43080 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Tk_mRNA_mods_rep1_AT_01-r001 Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_mRNA_mods_rep1_AT_01-r001.d SUBJECT_SAMPLE_FACTORS - Tk_mRNA_mods_rep1_AT_01-r002 Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_mRNA_mods_rep1_AT_01-r002.d SUBJECT_SAMPLE_FACTORS - Tk_mRNA_mods_rep1_AT_01-r003 Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_mRNA_mods_rep1_AT_01-r003.d SUBJECT_SAMPLE_FACTORS - Tk_mRNA_mods_rep2_AT_01-r001 Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_mRNA_mods_rep2_AT_01-r001.d SUBJECT_SAMPLE_FACTORS - Tk_mRNA_mods_rep2_AT_01-r002 Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_mRNA_mods_rep2_AT_01-r002.d SUBJECT_SAMPLE_FACTORS - Tk_mRNA_mods_rep2_AT_01-r003 Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_mRNA_mods_rep2_AT_01-r003.d SUBJECT_SAMPLE_FACTORS - TAM4_mRNA_rA_rep1-r001 Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_rRNA_depl_rep1_rA_quant-r001.d SUBJECT_SAMPLE_FACTORS - TAM4_mRNA_rA_rep1-r002 Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_rRNA_depl_rep1_rA_quant-r002.d SUBJECT_SAMPLE_FACTORS - TAM4_mRNA_rU_rep1-r001 Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_rRNA_depl_rep1_rU_quant-r001.d SUBJECT_SAMPLE_FACTORS - TAM4_mRNA_rU_rep1-r002 Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_rRNA_depl_rep1_rU_quant-r002.d SUBJECT_SAMPLE_FACTORS - TAM4_mRNA_GC_rep1-r001 Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_rRNA_depl_rep1_GC_quant-r001.d SUBJECT_SAMPLE_FACTORS - TAM4_mRNA_GC_rep1-r002 Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_rRNA_depl_rep1_GC_quant-r002.d SUBJECT_SAMPLE_FACTORS - TAM4_mRNA_rA_rep2 Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_rep2_rRNA_depl_rA_quant.d SUBJECT_SAMPLE_FACTORS - TAM4_mRNA_rU_rep2 Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_rep2_rRNA_depl_rU_quant.d SUBJECT_SAMPLE_FACTORS - TAM4_mRNA_GC_rep2 Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_rep2_rRNA_depl_GC_quant.d SUBJECT_SAMPLE_FACTORS - M_mari_mRNA_rA_rep1-r001 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_rRNA_depl_rep1_rA_quant-r001.d SUBJECT_SAMPLE_FACTORS - M_mari_mRNA_rA_rep1-r002 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_rRNA_depl_rep1_rA_quant-r002.d SUBJECT_SAMPLE_FACTORS - M_mari_mRNA_rU_rep1-r001 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_rRNA_depl_rep1_rU_quant-r001.d SUBJECT_SAMPLE_FACTORS - M_mari_mRNA_rU_rep1-r002 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_rRNA_depl_rep1_rU_quant-r002.d SUBJECT_SAMPLE_FACTORS - M_mari_mRNA_GC_rep1-r001 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_rRNA_depl_rep1_GC_quant-r001.d SUBJECT_SAMPLE_FACTORS - M_mari_mRNA_GC_rep1-r002 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_rRNA_depl_rep1_GC_quant-r002.d SUBJECT_SAMPLE_FACTORS - M_mari_mRNA_rA_rep2 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_mRNA_rA_Mods.d SUBJECT_SAMPLE_FACTORS - M_mari_mRNA_rU_rep2 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_mRNA_rU_Mods.d SUBJECT_SAMPLE_FACTORS - M_mari_mRNA_GC_rep2 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_mRNA_GC_Mods.d SUBJECT_SAMPLE_FACTORS - S_acid_mRNA_rA_rep1-r001 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rRNA_depl_rA_quant-r001.d SUBJECT_SAMPLE_FACTORS - S_acid_mRNA_rA_rep1 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rRNA_depl_rA_quant-r002.d SUBJECT_SAMPLE_FACTORS - S_acid_mRNA_rU_rep1-r001 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rep1_rU_quant_01-r001.d SUBJECT_SAMPLE_FACTORS - S_acid_mRNA_rU_rep1-r002 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rep1_rU_quant_01-r002.d SUBJECT_SAMPLE_FACTORS - S_acid_mRNA_rU_rep1-r003 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rep1_rU_quant_01-r003.d SUBJECT_SAMPLE_FACTORS - S_acid_mRNA_GC_rep1-r001 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rep1_rRNA_depl_GC_quant-r001.d SUBJECT_SAMPLE_FACTORS - S_acid_mRNA_GC_rep1-r002 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rep1_rRNA_depl_GC_quant-r002.d SUBJECT_SAMPLE_FACTORS - S_acid_mRNA_rA_rep2 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rRNA_depl_rA_quant_rep2-r001.d SUBJECT_SAMPLE_FACTORS - S_acid_mRNA_rU_rep2 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rRNA_depl_rep2_rU_quant.d SUBJECT_SAMPLE_FACTORS - S_acid_mRNA_GC_rep2 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rep2_rRNA_depl_GC_quant-r001.d SUBJECT_SAMPLE_FACTORS - S_island_mRNA_rA_rep1-r001 Species:Sulfolobus islandicus | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_island_rep1_rRNA_depl_rA_quant-r001.d SUBJECT_SAMPLE_FACTORS - S_island_mRNA_rA_rep1-r002 Species:Sulfolobus islandicus | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_island_rep1_rRNA_depl_rA_quant-r002.d SUBJECT_SAMPLE_FACTORS - S_island_mRNA_rU_rep1-r001 Species:Sulfolobus islandicus | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_island_rep1_rRNA_depl_rU_quant-r001.d SUBJECT_SAMPLE_FACTORS - S_island_mRNA_rU_rep1-r002 Species:Sulfolobus islandicus | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_island_rep1_rRNA_depl_rU_quant-r002.d SUBJECT_SAMPLE_FACTORS - S_island_mRNA_GC_rep1 Species:Sulfolobus islandicus | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_island_rep1_rRNA_depl_GC_quant-r001.d SUBJECT_SAMPLE_FACTORS - S_island_mRNA_rA_rep2 Species:Sulfolobus islandicus | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M16_4_mRNA_rA_Mods.d SUBJECT_SAMPLE_FACTORS - S_island_mRNA_rU_rep2 Species:Sulfolobus islandicus | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M16_4_mRNA_rU_Mods.d SUBJECT_SAMPLE_FACTORS - S_island_mRNA_GC_rep2 Species:Sulfolobus islandicus | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M16_4_mRNA_GC_Mods.d #COLLECTION CO:COLLECTION_SUMMARY T. kodakarensis strains were grown at 85C in anaerobic artificial sea water CO:COLLECTION_SUMMARY supplemented with yeast extract and tryptone to mid-exponential phase (Optical CO:COLLECTION_SUMMARY density ~0.3) before harvest. Sources of Thermococcus sp. AM4, Sulfolobus CO:COLLECTION_SUMMARY islandicus M16.4, Sulfolobus acidocaldarius, and Methanococcus maripaludis CO:COLLECTION_SUMMARY biomasses were contributed by C.S. Raman (University of Maryland), Rachel CO:COLLECTION_SUMMARY Whitaker (University of Illinois at Urbana-Champaign), Sonja-Verena Albers CO:COLLECTION_SUMMARY (University of Freiburg), and Barney Whitman (University of Georigia), CO:COLLECTION_SUMMARY respectively. Harvested archaeal cell pellets were resuspended in 10 mL of TRI CO:COLLECTION_SUMMARY reagent (Molecular Research Center, Inc., Cat #TR118). The resuspended cells CO:COLLECTION_SUMMARY were homogenized using a beads beater at 4.0 m/s for 20 seconds x 2 cycles (MP CO:COLLECTION_SUMMARY Biomedicals, FastPrep-24TM). Subsequently, the mixture was centrifuged at 14000 CO:COLLECTION_SUMMARY g for 5 minutes to precipitate any cell debris. Supernatants were collected CO:COLLECTION_SUMMARY post-centrifugation and treated with 50 μL of BAN reagent (Molecular Research CO:COLLECTION_SUMMARY Center, Inc., Cat #BN191) per mL of supernatant for aqueous-organic phase CO:COLLECTION_SUMMARY separation. RNA from the aqueous phase was isolated by isopropanol precipitation CO:COLLECTION_SUMMARY and subjected to DNase I treatment (NEB, Cat #M0303S) to remove genomic DNA CO:COLLECTION_SUMMARY contamination. To further purify the DNase I-treated RNA, an equal volume of CO:COLLECTION_SUMMARY acid phenol-chloroform with isoamyl alcohol (125:24:1, Thermo Fisher Scientific, CO:COLLECTION_SUMMARY Cat #AM9722) was added to the reaction and centrifuged at 21300 g for 2 minutes CO:COLLECTION_SUMMARY to separate the aqueous phase from the organic phase. The aqueous phase CO:COLLECTION_SUMMARY containing RNA was then precipitated with 1.5 volumes of isopropanol and 0.1 CO:COLLECTION_SUMMARY volume of sodium acetate (Sigma Aldrich, Cat #S7899) at –20°C overnight. CO:COLLECTION_SUMMARY Finally, the precipitated RNA pellets were washed with 75% ethanol and dissolved CO:COLLECTION_SUMMARY in nuclease-free water. CO:SAMPLE_TYPE Ribonucleic acid #TREATMENT TR:TREATMENT_SUMMARY To remove rRNA and tRNA, total RNA was separated into large (> 200 nt) and small TR:TREATMENT_SUMMARY RNA (< 200 nt) fractions using the RNA Clean and Concentrator Kit (Zymo TR:TREATMENT_SUMMARY Research, Cat #R1017). Subsequently, 50 ug of the large RNA fraction were TR:TREATMENT_SUMMARY subjected to rRNA depletion using the NEBNext rRNA Depletion Kit (NEB, Cat TR:TREATMENT_SUMMARY #E7850X) with the following changes: The NEBNext rRNA depletion solutions TR:TREATMENT_SUMMARY provided in the kit were substituted for customized DNA probe mixtures (at 1 uM TR:TREATMENT_SUMMARY for each probe) fully complementary to rRNA sequences corresponding to each TR:TREATMENT_SUMMARY archaeal species; All volumes for the probe hybridization, RNase H and DNase I TR:TREATMENT_SUMMARY digestion reactions were scaled up by fivefold in 10 parallel reactions. TR:TREATMENT_SUMMARY Following the enzymatic treatment, the reactions were cleaned up using RNA Clean TR:TREATMENT_SUMMARY and Concentrator Kit (Zymo Research, Cat #R1017), the mRNA-enriched fractions TR:TREATMENT_SUMMARY were eluted in 10 uL water and combined. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY mRNA-enriched samples were digested to nucleosides at 37°C overnight using a SP:SAMPLEPREP_SUMMARY Nucleoside Digestion Mix (NEB, Cat #M0649S). The digested RNAs were subsequently SP:SAMPLEPREP_SUMMARY injected without prior purification on an Agilent 1290 Infinity II UHPLC SP:SAMPLEPREP_SUMMARY equipped with a G7117 diode array detector and an Agilent 6495C SP:SAMPLEPREP_SUMMARY Triple-Quadrupole Mass Spectrometer operating in positive electrospray SP:SAMPLEPREP_SUMMARY ionization (+ESI) mode. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Solvent A pH is 4.5 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Waters XSelect HSS T3 XP (100 × 2.1mm, 2.5um) CH:SOLVENT_A 100% water; 10mM ammonium acetate CH:SOLVENT_B 100% methanol CH:FLOW_GRADIENT 1%-40% Solvent B in 7.5 min CH:FLOW_RATE 0.6mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass spectrometric data were acquired using dynamic multiple reaction monitoring MS:MS_COMMENTS (DMRM) mode. Identification of each nucleoside species was based on the MS:MS_COMMENTS associated retention time and mass transition in the extracted chromatogram. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS femtomole MS_METABOLITE_DATA_START Samples TAM4_mRNA_rA_rep1-r001 TAM4_mRNA_rA_rep1-r002 TAM4_mRNA_rA_rep2 M_mari_mRNA_rA_rep1-r001 M_mari_mRNA_rA_rep1-r002 M_mari_mRNA_rA_rep2 S_acid_mRNA_rA_rep1-r001 S_acid_mRNA_rA_rep1 S_acid_mRNA_rA_rep2 S_island_mRNA_rA_rep1-r001 S_island_mRNA_rA_rep1-r002 S_island_mRNA_rA_rep2 Factors Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Species:Methanococcus maripaludis | Sample source:rRNA_depletion Species:Methanococcus maripaludis | Sample source:rRNA_depletion Species:Methanococcus maripaludis | Sample source:rRNA_depletion Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Species:Sulfolobus islandicus | Sample source:rRNA_depletion Species:Sulfolobus islandicus | Sample source:rRNA_depletion Species:Sulfolobus islandicus | Sample source:rRNA_depletion adenosine 148.6849027 155.8994227 597.4855894 2065.564543 2052.653647 986.371136 1481.995121 1523.50112 1179.261985 1647.27643 1711.058245 668.0604583 1-methyladenosine 0.442675499 0.444625523 1.426461221 0.122176686 0.126553068 0.506059856 1.924892341 1.988094426 1.387345699 0.259877127 0.25392534 1.123263748 1-methylinosine 0.055360474 0.044045336 0.219154745 0.083594036 0.088108075 0.203664229 1.270753649 1.274607347 0.960573392 0.117443931 0.084978037 0.132649068 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z Precursor Ion m/z Product Ion adenosine 268.1 136.1 1-methyladenosine 282.1 150.1 1-methylinosine 283.1 151.1 METABOLITES_END #END