#METABOLOMICS WORKBENCH atsai_20250205_193220 DATATRACK_ID:5596 STUDY_ID:ST003727 ANALYSIS_ID:AN006113 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%-23% Solvent B in 7.5 min CH:FLOW_RATE 0.6 mL/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 Tk_mRNA_mods_rep1_AT_01-r001 Tk_mRNA_mods_rep1_AT_01-r002 Tk_mRNA_mods_rep1_AT_01-r003 Tk_mRNA_mods_rep2_AT_01-r001 Tk_mRNA_mods_rep2_AT_01-r002 Tk_mRNA_mods_rep2_AT_01-r003 TAM4_mRNA_GC_rep1-r001 TAM4_mRNA_GC_rep1-r002 TAM4_mRNA_rU_rep1-r001 TAM4_mRNA_rU_rep1-r002 TAM4_mRNA_GC_rep2 TAM4_mRNA_rU_rep2 M_mari_mRNA_GC_rep1-r001 M_mari_mRNA_GC_rep1-r002 M_mari_mRNA_rU_rep1-r001 M_mari_mRNA_rU_rep1-r002 M_mari_mRNA_GC_rep2 M_mari_mRNA_rU_rep2 S_acid_mRNA_GC_rep1-r001 S_acid_mRNA_GC_rep1-r002 S_acid_mRNA_rU_rep1-r001 S_acid_mRNA_rU_rep1-r002 S_acid_mRNA_rU_rep1-r003 S_acid_mRNA_GC_rep2 S_acid_mRNA_rU_rep2 S_island_mRNA_GC_rep1 S_island_mRNA_rU_rep1-r001 S_island_mRNA_rU_rep1-r002 S_island_mRNA_GC_rep2 S_island_mRNA_rU_rep2 Factors Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Species:Thermococcus kodakarensis | Sample source:rRNA_depletion 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: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: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 acidocaldarius | 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 Species:Sulfolobus islandicus | Sample source:rRNA_depletion Species:Sulfolobus islandicus | Sample source:rRNA_depletion guanosine 1765.589197 1804.82672 1834.193633 1173.992154 1150.833556 1164.193437 319.8849574 319.3764712 1132.64784 1743.171078 1777.452945 920.4377035 1357.345178 1368.519085 1395.003655 1612.317288 464.7091364 cytidine 1485.508803 1515.817531 1531.32304 1046.643226 1023.448638 1030.672424 261.6991244 258.3504933 899.615868 1891.170092 1921.372566 360.5549796 1094.782206 1113.472958 1143.520528 1527.88004 304.599905 uridine 1239.404403 1287.639281 1301.371839 789.8925478 754.5782071 767.4816916 660.1442606 410.266996 198.4370334 2070.689995 2060.523258 793.2385085 1578.427081 1589.476929 1618.580187 1837.929235 2017.066257 2013.795292 597.6397564 adenosine 1389.151752 1415.334015 1432.660316 1112.417488 1172.359939 1113.927243 1-methyladenosine 3.439977984 3.468036913 3.466929095 3.09468295 3.074938491 3.087085247 5-methylcytidine 2.899887486 3.047517395 2.83787459 2.057343583 2.012292711 2.097703247 0.665100919 0.664226581 4.152294766 0.70308407 0.677638876 0.451272293 3.892004055 4.157112529 4.055959484 0.953019662 1.499706414 2'-O-methylcytidine 3.031244791 3.010914784 3.329688169 2.759327924 2.64696561 2.595999216 0.349631688 0.384054448 3.293913239 1.143764042 1.240603165 0.720556799 6.130548011 6.134065035 5.754220896 1.904110611 5.57588796 1-methylinosine 0.380208183 0.337947276 0.423280287 0.164396264 0.131511095 0.149097443 2'-O-methylguanosine 5.318514258 5.378438437 5.51987645 4.301257592 3.938759121 4.226058801 0.331342703 0.277141594 3.882941098 0.091423648 0.408333864 0.201253183 2.57442105 2.77371004 2.574719406 1.569160774 3.01262596 N4-acetylcytidine 3.478314597 3.610816733 3.81399251 1.859573518 1.655004454 1.796493115 1.651709074 1.683786389 7.961088991 0.14334826 0.114088142 0 0.042916022 0.045367431 1.176175739 0.119976158 1.18250035 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z Precursor Ion m/z Product ion guanosine 284.1 152.1 cytidine 244.1 112.1 uridine 245.11 113 adenosine 268.1 136.1 1-methyladenosine 282.1 150.1 5-methylcytidine 258.1 126.1 2'-O-methylcytidine 258.1 112.1 1-methylinosine 283.1 151.1 2'-O-methylguanosine 298.1 152.1 N4-acetylcytidine 286.11 154.1 METABOLITES_END #END