#METABOLOMICS WORKBENCH jad2033_20221115_170125 DATATRACK_ID:3575 STUDY_ID:ST002352 ANALYSIS_ID:AN004097 PROJECT_ID:PR001509 VERSION 1 CREATED_ON February 28, 2023, 11:03 am #PROJECT PR:PROJECT_TITLE Biomolecular condensates create phospholipid-enriched microenvironments PR:PROJECT_TYPE Metabolomics of in vitro condensates PR:PROJECT_SUMMARY Proteins and RNA are able to phase separate from the aqueous cellular PR:PROJECT_SUMMARY environment to form sub-cellular compartments called condensates. This process PR:PROJECT_SUMMARY results in a protein-RNA mixture that is chemically distinct from the PR:PROJECT_SUMMARY surrounding aqueous phase. Here we use mass spectrometry to characterize the PR:PROJECT_SUMMARY metabolomes of condensates. To test this, we prepared mixtures of PR:PROJECT_SUMMARY phase-separated proteins and cellular metabolites and identified metabolites PR:PROJECT_SUMMARY enriched in the condensate phase. These proteins included SARS-CoV-2 PR:PROJECT_SUMMARY nucleocapsid, as well as low complexity domains of MED1 and HNRNPA1. PR:INSTITUTE Cornell University PR:DEPARTMENT Department of Pharmacology PR:LABORATORY Dr. Samie Jaffrey PR:LAST_NAME Dumelie PR:FIRST_NAME Jason PR:ADDRESS 1300 York Ave, LC-524, New York City, NY PR:EMAIL jdumes98@gmail.com PR:PHONE 6465690174 PR:FUNDING_SOURCE This work was supported by the National Institutes of Health grants R35NS111631 PR:FUNDING_SOURCE and R01CA186702 (S.R.J.); R01AR076029, R21ES032347 and R21NS118633 (Q.C.); and PR:FUNDING_SOURCE NIH P01 HD067244 and support from the Starr Cancer Consortium I13-0037 (S.S.G.). PR:PUBLICATIONS Under revision PR:CONTRIBUTORS Jason G. Dumelie, Qiuying Chen, Dawson Miller, Nabeel Attarwala, Steven S. Gross PR:CONTRIBUTORS and Samie R. Jaffrey1 #STUDY ST:STUDY_TITLE Biomolecular condensates create phospholipid-enriched microenvironments (Part 2) ST:STUDY_SUMMARY Proteins and RNA are able to phase separate from the aqueous cellular ST:STUDY_SUMMARY environment to form sub-cellular compartments called condensates. This process ST:STUDY_SUMMARY results in a protein-RNA mixture that is chemically distinct from the ST:STUDY_SUMMARY surrounding aqueous phase. Here we use mass spectrometry to characterize the ST:STUDY_SUMMARY metabolomes of condensates. To test this, we prepared mixtures of ST:STUDY_SUMMARY phase-separated proteins and cellular metabolites and identified metabolites ST:STUDY_SUMMARY enriched in the condensate phase. These proteins included SARS-CoV-2 ST:STUDY_SUMMARY nucleocapsid, as well as low complexity domains of MED1 and HNRNPA1. ST:INSTITUTE Cornell University ST:DEPARTMENT Department of Pharmacology ST:LABORATORY Dr. Samie Jaffrey ST:LAST_NAME Dumelie ST:FIRST_NAME Jason ST:ADDRESS 1300 York Ave, LC-524, New York City, NY ST:EMAIL jdumes98@gmail.com ST:STUDY_TYPE Metabolomes of in vitro synthesized condensates ST:PHONE 6465690174 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - MED1 Condensate Sample 4 Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:No heat step | timing of metabolite extract addition:standard source=Mouse liver metabolites; RAW_FILE_NAME=51_B_No650.mzdata.xml;p_51_B_No650.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Condensate Sample 5 Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:No heat step | timing of metabolite extract addition:standard source=Mouse liver metabolites; RAW_FILE_NAME=52_B.mzdata.xml;p_52_B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Condensate Sample 6 Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:No heat step | timing of metabolite extract addition:standard source=Mouse liver metabolites; RAW_FILE_NAME=53_B.mzdata.xml;p_53_B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Condensate Sample 7 Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation source=Mouse liver metabolites; RAW_FILE_NAME=61_B_No650.mzdata.xml;p_61_B_No650.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Condensate Sample 8 Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation source=Mouse liver metabolites; RAW_FILE_NAME=62_B.mzdata.xml;p_62_B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Condensate Sample 9 Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation source=Mouse liver metabolites; RAW_FILE_NAME=63_B.mzdata.xml;p_63_B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 4 Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:No heat step | timing of metabolite extract addition:standard source=Mouse liver metabolites; RAW_FILE_NAME=51_I_No650.mzdata.xml;p_51_I_No650.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 5 Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:No heat step | timing of metabolite extract addition:standard source=Mouse liver metabolites; RAW_FILE_NAME=52_I.mzdata.xml;p_52_I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 6 Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:No heat step | timing of metabolite extract addition:standard source=Mouse liver metabolites; RAW_FILE_NAME=53_I.mzdata.xml;p_53_I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 7 Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation source=Mouse liver metabolites; RAW_FILE_NAME=61_I_No650.mzdata.xml;p_61_I_No650.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 8 Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation source=Mouse liver metabolites; RAW_FILE_NAME=62_I.mzdata.xml;p_62_I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 9 Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation source=Mouse liver metabolites; RAW_FILE_NAME=63_I.mzdata.xml;p_63_I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Aqueous Sample 4 Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:No heat step | timing of metabolite extract addition:standard source=Mouse liver metabolites; RAW_FILE_NAME=51_T_No650.mzdata.xml;p_51_T_No650.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Aqueous Sample 5 Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:No heat step | timing of metabolite extract addition:standard source=Mouse liver metabolites; RAW_FILE_NAME=52_T.mzdata.xml;p_52_T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Aqueous Sample 6 Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:No heat step | timing of metabolite extract addition:standard source=Mouse liver metabolites; RAW_FILE_NAME=53_T.mzdata.xml;p_53_T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Aqueous Sample 7 Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation source=Mouse liver metabolites; RAW_FILE_NAME=61_T_No650.mzdata.xml;p_61_T_No650.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Aqueous Sample 8 Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation source=Mouse liver metabolites; RAW_FILE_NAME=62_T.mzdata.xml;p_62_T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Aqueous Sample 9 Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation source=Mouse liver metabolites; RAW_FILE_NAME=63_T.mzdata.xml;p_63_T.mzdata.xml #COLLECTION CO:COLLECTION_SUMMARY Condensate metabolomics. Mouse metabolites were collected from the liver of CO:COLLECTION_SUMMARY female mice using methanol extraction. After euthanizing a mouse, the liver was CO:COLLECTION_SUMMARY immediately frozen in liquid nitrogen. We then used cold 80% methanol to extract CO:COLLECTION_SUMMARY metabolites. This method effectively quenches metabolic activity and is CO:COLLECTION_SUMMARY well-established for extracting a broad range of metabolites, including polar CO:COLLECTION_SUMMARY metabolites77,78. First, 1 ml of 80% methanol was added to the liver and CO:COLLECTION_SUMMARY incubated for 10 min at -20oC. Glass beads were added to the liver and then the CO:COLLECTION_SUMMARY liver was lysed by bead-beating for 45 s using a Tissuelyser cell disrupter CO:COLLECTION_SUMMARY (Qiagen). The lysate was incubated for 10 min at -20oC and centrifuged (13200 CO:COLLECTION_SUMMARY rpm, 5 min) to separate metabolites from macromolecules. The supernatant was CO:COLLECTION_SUMMARY collected and 200 µl of 80% methanol was added to the pellet. The incubation, CO:COLLECTION_SUMMARY shaking and centrifugation steps were repeated twice to extract more metabolites CO:COLLECTION_SUMMARY from the pellet. The three supernatants were combined and centrifuged (14000 CO:COLLECTION_SUMMARY rpm, 10 min) to separate any remaining macromolecules from the metabolites. The CO:COLLECTION_SUMMARY combined supernatants were dried using a SpeedVac Concentrator (Savant, CO:COLLECTION_SUMMARY SPD131DDA) at 25oC and the dried metabolite samples were stored at -80oC. The CO:COLLECTION_SUMMARY amount of protein in the pellet was measured using the Quick Start Bradford CO:COLLECTION_SUMMARY assay to calculate the metabolites’ protein equivalent mass. Mouse metabolites CO:COLLECTION_SUMMARY were initially re-suspended in condensate buffer (50 mM NH4HCO3 pH 7.5, 50 mM CO:COLLECTION_SUMMARY NaCl, 1 mM DTT) to a protein equivalent concentration of 938 g/l. The chosen CO:COLLECTION_SUMMARY final concentration of metabolites is slightly lower than the 200-300 g/l CO:COLLECTION_SUMMARY protein concentration observed in cells79. Metabolites that were not fully CO:COLLECTION_SUMMARY soluble in condensate buffer were removed by centrifugation (2x5 min, 16,000 g CO:COLLECTION_SUMMARY each), in which only the supernatant was retained. Due to the lack of crowding CO:COLLECTION_SUMMARY agents, phase separation required greater concentrations of protein and RNA than CO:COLLECTION_SUMMARY typically employed for nucleocapsid and MED1 condensate formation17,32. Purified CO:COLLECTION_SUMMARY protein (37.5 μM) was centrifuged (1 min, 1,000 g) to disrupt any existing CO:COLLECTION_SUMMARY condensates and to remove any precipitated proteins. Purified protein (final CO:COLLECTION_SUMMARY concentration, 30 μM) was combined with metabolites (final concentration, 150 CO:COLLECTION_SUMMARY g/l protein equivalent) and then phage lambda RNA (final concentration, 0.15 CO:COLLECTION_SUMMARY μM) in a total volume of 300 µl. An input sample (10 µl) was saved and then CO:COLLECTION_SUMMARY the sample was allowed to incubate for 10 min at 25oC. Condensates were then CO:COLLECTION_SUMMARY separated from the aqueous environment by centrifugation (10 min, 12,500 g, CO:COLLECTION_SUMMARY 25oC). The aqueous phase was removed from the condensate phase and then equal CO:COLLECTION_SUMMARY volumes (usually ~ 2 µl) of the aqueous fraction, condensate fraction and input CO:COLLECTION_SUMMARY sample were processed for metabolomics using identical approaches as described CO:COLLECTION_SUMMARY below. First the samples were diluted in ammonium bicarbonate buffer (50 mM CO:COLLECTION_SUMMARY NH4HCO3 pH 7.5) and briefly heated (2 min, 65oC) to disrupt condensates before CO:COLLECTION_SUMMARY being added immediately to 4x volume of ice-cold 100% methanol to precipitate CO:COLLECTION_SUMMARY protein and RNA. This heating step does not appear to be necessary for CO:COLLECTION_SUMMARY extracting these metabolites and can be excluded (Extended Data Fig. 2e,g). CO:COLLECTION_SUMMARY Protein and RNA were separated from metabolites by vortexing the samples (2 CO:COLLECTION_SUMMARY min), followed by incubation at -25oC (10 min) and then centrifugation (5 min, CO:COLLECTION_SUMMARY 13,000 rpm). The supernatant was saved and the process was repeated on the CO:COLLECTION_SUMMARY pellet two more times after adding 200 µl of 80% methanol each time to the CO:COLLECTION_SUMMARY pellet. The three supernatants were combined and centrifuged (10 min, 14000 rpm) CO:COLLECTION_SUMMARY to remove any additional macromolecules. The final supernatant was collected and CO:COLLECTION_SUMMARY dried using a SpeedVac Concentrator run at 25oC. Notably, in one subset of CO:COLLECTION_SUMMARY experiments, metabolites were added to MED1 condensates after the 10 min CO:COLLECTION_SUMMARY incubation rather than prior to the incubation. In another, the heat step CO:COLLECTION_SUMMARY immediately after fractions were collected was removed. CO:SAMPLE_TYPE Liver CO:COLLECTION_METHOD 80% methanol CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Mouse liver metabolites were combined with either the condensate-forming TR:TREATMENT_SUMMARY low-complexity domains of HNRNPA1, MED1 or full-length SARS-CoV-2 nucleocapsid. TR:TREATMENT_SUMMARY Condensates were stimulated with either 0 nM, 150 nM or 600 nM RNA. Condensates TR:TREATMENT_SUMMARY were centrifuged to the bottom of a 600 ul tube. Equal fractions from the input TR:TREATMENT_SUMMARY sample, aqueous phase and condensate phases were collected separately. TR:TREATMENT_SUMMARY Metabolites were extracted from each fraction. Alternatively, liver metabolites TR:TREATMENT_SUMMARY were added 10 min after stimulating condensate formation with phage RNA and TR:TREATMENT_SUMMARY incubating for 2 min prior to centrifugation. In a separate set of experiments, TR:TREATMENT_SUMMARY the extraction procedure from the three fractions (ie aqueous, condensate and TR:TREATMENT_SUMMARY input) was altered to remove a heat step. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from each fraction and the input for LC-MS as SP:SAMPLEPREP_SUMMARY follows. First the samples were diluted in ammonium bicarbonate buffer (50 mM SP:SAMPLEPREP_SUMMARY NH4HCO3 pH 7.5) and briefly heated (2 min, 65oC) to disrupt condensates before SP:SAMPLEPREP_SUMMARY being added immediately to 4x volume of ice-cold 100% methanol to precipitate SP:SAMPLEPREP_SUMMARY protein and RNA. This heating step does not appear to be necessary for SP:SAMPLEPREP_SUMMARY extracting these metabolites and can be excluded (Extended Data Fig. 2e,g). SP:SAMPLEPREP_SUMMARY Protein and RNA were separated from metabolites by vortexing the samples (2 SP:SAMPLEPREP_SUMMARY min), followed by incubation at -25oC (10 min) and then centrifugation (5 min, SP:SAMPLEPREP_SUMMARY 13,000 rpm). The supernatant was saved and the process was repeated on the SP:SAMPLEPREP_SUMMARY pellet two more times after adding 200 µl of 80% methanol each time to the SP:SAMPLEPREP_SUMMARY pellet. The three supernatants were combined and centrifuged (10 min, 14000 rpm) SP:SAMPLEPREP_SUMMARY to remove any additional macromolecules. The final supernatant was collected and SP:SAMPLEPREP_SUMMARY dried using a SpeedVac Concentrator run at 25oC. On the day of metabolite SP:SAMPLEPREP_SUMMARY analysis, dried-down extracts were reconstituted in 150 µl 70% acetonitrile, at SP:SAMPLEPREP_SUMMARY a relative protein concentration of ~ 2 µg/µl, and 4 µl of this reconstituted SP:SAMPLEPREP_SUMMARY extract was injected for LC/MS-based targeted and untargeted metabolite SP:SAMPLEPREP_SUMMARY profiling. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Tissue extracts were analyzed by LC/MS as described previously, using a platform CH:CHROMATOGRAPHY_SUMMARY comprised of an Agilent Model 1290 Infinity II liquid chromatography system CH:CHROMATOGRAPHY_SUMMARY coupled to an Agilent 6550 iFunnel time-of-flight MS analyzer. Chromatography of CH:CHROMATOGRAPHY_SUMMARY metabolites utilized aqueous normal phase (ANP) chromatography on a Diamond CH:CHROMATOGRAPHY_SUMMARY Hydride column (Microsolv). Mobile phases consisted of: (A) 50% isopropanol, CH:CHROMATOGRAPHY_SUMMARY containing 0.025% acetic acid, and (B) 90% acetonitrile containing 5 mM ammonium CH:CHROMATOGRAPHY_SUMMARY acetate. To eliminate the interference of metal ions on chromatographic peak CH:CHROMATOGRAPHY_SUMMARY integrity and electrospray ionization, EDTA was added to the mobile phase at a CH:CHROMATOGRAPHY_SUMMARY final concentration of 5 µM. The following gradient was applied: 0-1.0 min, 99% CH:CHROMATOGRAPHY_SUMMARY B; 1.0-15.0 min, to 20% B; 15.0 to 29.0, 0% B; 29.1 to 37min, 99% B. CH:CHROMATOGRAPHY_TYPE Normal phase CH:INSTRUMENT_NAME Agilent Model 1290 Infinity II liquid chromatography system CH:COLUMN_NAME Cogent Diamond Hydride (150 × 2.1 mm, 4um) CH:SOLVENT_A 50% isopropanol, containing 0.025% acetic acid CH:SOLVENT_B 90% acetonitrile containing 5 mM ammonium acetate #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE Other MS:ION_MODE POSITIVE MS:MS_COMMENTS LC/MS-based targeted and untargeted metabolite profiling. For targeted analysis, MS:MS_COMMENTS raw LC/MS data was extracted by MassProfinder 8.0 (Agilent Technologies) using MS:MS_COMMENTS an in-house annotated personal metabolite database that contains 863 metabolites MS:MS_COMMENTS (Agilent Technologies). Additionally, molecular feature extraction (MFE) was MS:MS_COMMENTS performed for untargeted metabolite profiling using MassProfinder 8.0 (Agilent MS:MS_COMMENTS Technologies). The untargeted molecular features were imported into MassProfiler MS:MS_COMMENTS Professional 15.1 (MPP, Agilent Technologies) and searched against Metlin MS:MS_COMMENTS personal metabolite database (PCDL database 8.0), Human Metabolome Database MS:MS_COMMENTS (HMDB) and an in-house phospholipid database for tentative metabolite ID MS:MS_COMMENTS assignments, based on monoisotopic neutral mass (< 5 ppm mass accuracy) matches. MS:MS_COMMENTS Furthermore, a molecular formula generator (MFG) algorithm in MPP was used to MS:MS_COMMENTS generate and score empirical molecular formulae, based on a weighted MS:MS_COMMENTS consideration of monoisotopic mass accuracy, isotope abundance ratios, and MS:MS_COMMENTS spacing between isotope peaks. A tentative compound ID was assigned when PCDL MS:MS_COMMENTS database and MFG scores concurred for a given candidate molecule. Tentatively MS:MS_COMMENTS assigned molecules were reextracted using Profinder 8.0 for confirmation of MS:MS_COMMENTS untargeted results. For phospholipids, assignment of IDs was based on the MS:MS_COMMENTS defined pattern of neutral loss and head group fragment ions. Metabolites from MS:MS_COMMENTS targeted and untargeted extraction were combined for further statistical MS:MS_COMMENTS analysis among groups of input, aqueous and condensate fractions. Metabolites MS:MS_COMMENTS were removed from our analysis if they had a low ion count or high variation in MS:MS_COMMENTS input samples. Measurements of metabolite ion counts in input samples should be MS:MS_COMMENTS replicates across experiments. As such, differences in metabolite ion counts MS:MS_COMMENTS reflect experimental variability. To determine appropriate cut-offs, we examined MS:MS_COMMENTS the relationship between metabolite ion counts and their variation across input MS:MS_COMMENTS sample technical replicates. Metabolites with a median of < 1000 ion MS:MS_COMMENTS counts/sample tended to have high variation across samples. As a result, these MS:MS_COMMENTS metabolites were removed. Metabolites were also removed with > 2.5 standard MS:MS_COMMENTS deviation in log2(ion counts) since the input measurements for these metabolites MS:MS_COMMENTS were particularly unreliable relative to what was observed for other MS:MS_COMMENTS metabolites. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Ion counts MS_METABOLITE_DATA_START Samples MED1 Condensate Sample 4 MED1 Condensate Sample 5 MED1 Condensate Sample 6 MED1 Condensate Sample 7 MED1 Condensate Sample 8 MED1 Condensate Sample 9 MED1 Input Sample 4 MED1 Input Sample 5 MED1 Input Sample 6 MED1 Input Sample 7 MED1 Input Sample 8 MED1 Input Sample 9 MED1 Aqueous Sample 4 MED1 Aqueous Sample 5 MED1 Aqueous Sample 6 MED1 Aqueous Sample 7 MED1 Aqueous Sample 8 MED1 Aqueous Sample 9 Factors Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:No heat step | timing of metabolite extract addition:standard Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:No heat step | timing of metabolite extract addition:standard Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:No heat step | timing of metabolite extract addition:standard Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation Protein:MED1 | RNA:150 nM | fraction:condensate | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:No heat step | timing of metabolite extract addition:standard Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:No heat step | timing of metabolite extract addition:standard Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:No heat step | timing of metabolite extract addition:standard Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation Protein:MED1 | RNA:150 nM | fraction:input | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:No heat step | timing of metabolite extract addition:standard Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:No heat step | timing of metabolite extract addition:standard Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:No heat step | timing of metabolite extract addition:standard Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation Protein:MED1 | RNA:150 nM | fraction:aqueous | Extraction from condensate:standard | timing of metabolite extract addition:Metabolites added after 10 min incubation 1-Methyladenosine 931477.939358662 625913.439959874 684035.532908752 762981.418315235 830815.756756586 1022031.10532864 1408442.50700716 1443670.83297441 1679662.33353601 1455828.04395749 1571596.76354959 1460995.86344118 1233558.55433781 1385362.58927198 1411066.48091163 1419516.96919366 1541054.02786853 1236252.35471872 1-Methylnicotinamide 171046.863368528 145439.512801363 128954.542374116 151503.258974001 124916.612885133 212949.713470858 167177.620170595 187956.995074612 203546.194543378 170223.912221071 183981.010936891 196633.660935058 141667.412874147 187170.851538389 181695.427853391 170206.8025 181503.706948242 178664.036932828 3-Methylhistamine 116997.126480179 106262.88744957 106940.830585175 109240.24806099 108769.913493577 112495.601834443 118335.871772385 109851.617338791 120433.236111526 117677.829234047 115287.396785278 119687.610188369 93519.9057958985 107121.317806625 120345.719043808 97315.1835235596 111619.712806244 120875.839620743 3-Methylhistidine 176698.793208801 133541.329456115 137748.126945495 116632.548810303 88145.0462094116 155150.518064759 163078.655556274 210173.935996765 209063.619652466 167133.055782043 202000.715525635 184697.409146545 156914.152200806 201278.835097107 171423.462884277 192954.312989258 190204.273863281 161926.841324768 CAR 16:1;O 79717.7039508055 73786.0015313722 60623.8375073239 53998.9261767576 37520.1138049315 45957.1496417236 135 2078.90755859378 135 135 135 135 135 135 135 135 135 135 441.355@7.97, provisional ID = CAR 18:1-0H 66869.8035961914 74799.6972338865 61273.0953393552 58557.0578728024 32945.607158203 81225.1534649663 135 135 959.740140624997 981.988876953122 135 135 135 135 135 135 19568.1702731933 135 CAR 16:0;O 141391.064195434 122759.770332214 110982.923194885 103130.102100525 68484.4653124999 101253.650291565 902.169265624998 4979.40995672613 135 877.893335815362 135 5545.36759442131 8890.00319165023 3485.14504309073 3208.30737731938 3582.61218688956 25233.390395813 2444.20616345217 367.2674@7.75, provisional ID = CAR 14:2 19290.9184014892 16651.0401989746 23190.2507537841 22370.6526861571 19154.2729687501 16995.5351506347 20217.2848561403 19782.6844628907 14147.0311514282 19570.3623980714 43529.3481387327 12038.4885266113 17399.8282620242 24660.91815979 16509.2046554565 27445.2402471925 25440.6597373963 18939.1302172854 4-Aminobutyric Acid 310568.59515747 224687.842151733 261098.189969849 291940.573856811 276150.035570496 433910.531703614 299235.897903931 376113.413100281 368664.677114075 317648.437246582 368274.777950928 350971.870090209 394153.248054382 365201.469940368 388468.085776672 417484.220687805 420166.639949035 374644.062289734 4-Guanidinobutyric Acid 535964.681900331 444988.687590943 398710.577890564 468157.072652985 412644.748897766 710881.229955201 508225.77311554 579155.814190429 606005.647397705 543295.727809296 603055.378867005 595079.727483522 462581.953279113 582122.328800721 546527.754038299 557817.446863069 571677.394389052 556480.217046417 4-Imidazoleacetic Acid 2184884.97396734 2199415.73599126 2370435.38630128 2136851.48665576 2149472.58433027 3132146.69891271 2001118.60093006 2781039.22173131 2583448.07370825 2266742.39853442 2481917.920451 2199056.09358318 2696783.23736677 2386395.54544268 2123591.94497382 2352792.55683837 2402012.03607538 1991283.35498589 5-Aminopentanoic Acid 3845269.12459375 3448708.77702344 3236496.71391406 3430104.82867969 3328644.2471914 5540721.99380859 4130932.47046485 4601276.52269922 5388754.619875 4443151.81124219 4954697.41439062 4969033.33432031 3597957.40619141 4703286.77982422 4720111.82451953 4371320.94082421 5001365.50236329 4688618.80660547 5'-Methylthioadenosine 5976370.52675391 3698732.39720514 3403895.39897107 4917518.8189513 4124634.93122467 6192186.14522888 5809289.7378457 5345769.85555237 4981270.99289514 5127696.2770321 6269362.82148395 5893150.13097204 4602136.70028015 5063598.35787714 4643850.98719074 5018277.3176012 6701424.67598657 4436586.20806091 CAR 2:0 44998568.6468121 37539013.4688443 31967487.932435 38128520.82217 34095512.2384217 63579589.9691864 48130267.2002621 53679812.3283461 63072518.4490427 51298993.3031281 58815692.0738998 61443499.3426258 39749830.8800113 55317865.8857509 55456766.493017 54536853.2351546 58578134.6164189 53162649.4889729 Adenine 7397410.70291583 4073394.39676686 4086560.58920803 5820840.50327875 5963080.71137655 9449247.05688568 3313107.972708 2995695.24699184 4703274.04211445 3826489.98297997 4622088.49673657 5005687.32969623 4502202.31046751 4150564.83178388 5062336.92304498 4328031.57790018 5856725.98597261 4751243.37634445 Adenosine 5336356.25663555 3831025.82659137 3801380.45324937 3210447.6395713 3390443.442013 6760256.69795247 4485544.0524886 7747101.37612769 4203325.42249503 2370156.99220593 2932729.53108359 4467597.54947846 6196828.18061484 3795407.47013756 2995795.18721842 4866381.01681998 2418699.81751672 3988443.36306409 Alanine 3423686.03703856 2791518.73671778 3117595.7026493 2982021.9326728 2526796.74817376 4803371.18516986 3396932.4153781 4840282.48842437 4399227.60946966 3499356.88886956 4550138.68113405 4144089.68967096 4070741.39619978 4199588.2156184 3873628.09536323 4457256.79000159 4142860.79619624 3410965.00877483 323.2824@1.13, provisional ID = NAE 18:2 299156.199581909 332534.081146912 225521.377707214 286246.802763367 187069.372664612 188120.920386841 162128.952902039 182589.758862427 229421.655063477 195571.477509521 315652.685057007 132172.693394165 185427.423659485 202860.669422974 107097.135130737 192563.639896362 216706.002521301 283007.955654358 PC O-36:4 220951.59595581 250005.411711975 229062.265349121 222430.308404236 177062.677336121 277702.112716186 36880.7041607665 29568.4033468627 28818.6380493163 10396.4946850585 21406.9006570435 33756.2511602782 14178.0313897705 23302.3402532959 9191.96569366454 7523.23617034914 15660.7305563355 135 Arginine 19545.5717967987 22902.3486387635 15489.9046717836 18855.2810803987 23629.7449768829 12644.3822744751 13038.0691151428 22170.361473236 12268.4991294859 17142.7989910889 19304.7592453002 14947.6952703858 17854.3012752533 18109.9936334228 23817.1830300902 18216.5594439697 12765.5651675415 18106.3336685179 Betaine 232502228.924032 212439593.314385 198789071.243919 218613408.987552 196291904.245083 277059529.251174 232005252.124888 252124444.944557 254275824.598979 241422767.471075 246564438.806079 259987760.714632 227415524.579074 255497867.540984 230476448.030729 252833886.639622 250889621.387457 235383543.974227 Bilirubin 31323.7704082716 20150.5752766113 28414.4873063778 18801.755350647 14472.0384865723 21027.0229963485 2895.57053724372 4595.53069717407 2764.39262218607 3915.42802273559 1908.47737625122 5330.70562426758 4657.55705652535 1852.81467763579 1503.70783035278 2227.12326454163 635.478777160645 4203.05049362183 CAR 4:0 22506594.2850093 19055077.0810879 16673773.0249196 19824662.3274583 16999371.0677557 32360548.5327903 24229324.4663827 27367594.0444387 31068340.569064 25415069.5607868 30197410.0948337 31210377.253885 20483535.6614768 28047163.484138 27960007.5662794 28141147.1671774 29350985.7911464 26651756.9294919 538.3788@7.87, provisional ID = PC O-20:0 49971.244383545 48248.3009722901 46708.8782257079 39195.3828082276 28560.6459613038 54578.8526983643 135 135 135 135 135 135 135 135 135 135 135 135 Carnitine 70182975.8838861 56910081.0156781 51249384.2183645 60149008.8426079 51327819.994905 97387770.4603613 71922758.2546426 82485121.404552 94785579.6476801 77429679.509774 90324801.7891756 91580931.5885088 63059463.0217242 85615000.6397674 84651198.3811956 83114309.4109303 88440756.2929096 80906735.2883983 Choline 7949501.0381899 5756376.74437766 5232215.08186804 6162502.71525533 5614912.40628476 10567215.3405541 7176576.68574412 7594765.21201743 8839729.95724134 7578358.92024921 8586403.01379033 8943745.48578632 6247865.22593424 8201777.11735765 8304978.55275273 9230769.39645587 8864772.88560733 8279019.71306581 Citrulline 304334.209835266 300038.936507934 304497.604175537 299001.35632843 312492.796786377 305080.814596497 297474.055663452 294453.450983521 308466.662202453 302903.548554993 303191.860837158 290184.416964356 291028.010928528 300191.520861328 298884.776214295 287903.139903625 286693.746260987 278761.25694336 ST 21:4;O5 239067.380078737 190950.992342752 186521.700325472 177511.797678467 155405.599296722 242325.555876091 146801.723654156 156455.035597229 145846.020748947 154288.473302281 162615.765636537 155032.400800227 147036.384068105 146989.840725605 145749.734871046 166813.265883504 157160.969691737 6682.84040069578 Creatinine 2828778.06493994 2428452.38752099 2288432.93041443 2590676.64065503 2624024.95752734 3572842.68804065 2976542.49628051 3345643.43411669 3446529.09131324 3135975.38471386 3555858.60684131 3460330.49694457 2930175.51182691 3281895.31928759 3162759.61268335 3673317.60821764 3513032.22670167 2969884.50518384 Cytidine 4827.52072692871 7795.52582641606 4561.8287677002 8365.28261535649 13853.1279370118 29286.450355835 14940.295378418 15584.5667218017 42287.275460205 18145.7023101806 42120.8245355225 33673.1867126465 9407.88703308104 20139.9059545898 11148.919451294 75904.8579593506 21642.1305474853 26277.7968566895 Deoxycarnitine 1275758.28760484 1092471.23350188 932398.297070777 1117511.23324697 979241.327066989 1878878.232805 1361312.63409283 1553511.89743221 1761808.70901956 1463286.21785648 1676826.19863423 1698739.77773284 1181227.21814097 1577348.84503044 1587975.88292331 1541839.0023677 1655956.21875511 1494465.79764057 526.4679@1.14, provisional ID = DG(29:0) 3022.54074523926 2660.74808349609 5643.20452575684 5975.2662878418 5001.11675506591 135 4320.96093933105 5664.20416351318 7388.97519470215 12952.2657818604 1466.43268280029 6604.84252502442 5045.9336062622 2733.71379516602 1300.32292999268 1707.30632659912 7167.92901611328 135 CAR 12:0 25304.3635251466 27471.3296791993 19169.0626614989 17895.4720874023 12149.5688642578 23411.1384619139 135 135 135 135 135 135 135 135 135 135 135 135 Guanidinosuccinic Acid 320039.309605715 229114.950133058 240121.04602356 276221.799777833 253641.565548095 416416.659864503 341656.572406615 389917.240374757 385962.737041014 329180.132351074 385445.183183595 387862.264763186 320576.496838379 354086.024759521 349327.016336669 381207.74126465 397183.085234376 308340.427149658 Guanine 533381.396314927 415800.782060337 399132.849085022 436467.295093763 474905.572342371 707285.488128737 542736.377250961 497351.068965016 649048.709531288 530100.73520607 653523.969822861 653328.002333972 458075.171075073 511750.465349224 597259.444705044 544933.217757317 649131.265015595 561230.084080238 Guanosine 1161499.27260814 827065.687943006 794179.268959877 949503.520235895 984508.012919481 1594104.71217667 1073775.94195333 1112129.8858206 1383289.69846984 1118392.03948962 1348905.13412515 1373260.8201393 1047568.30746277 1077202.0751724 1187144.31338388 1110117.37726598 1374784.15519936 1152247.76124837 CAR 16:1 590957.742818605 599180.903480531 532083.253559876 487069.01722992 371143.574397583 677246.029265443 26297.7699499513 60477.1903015138 42253.0778259276 30463.5880676266 37799.5953594973 35857.3690563966 25554.0283471682 31748.2442300414 28077.8698272705 46158.103847351 35981.4860760498 25249.2405868529 CAR 6:0 5168684.27428533 4305514.73336047 4225292.22817889 4546881.62414142 3864415.26445166 7318367.00797735 5358195.00315533 6488788.84033997 6682456.89600684 5469011.6708515 6787383.20261372 6513634.7946856 5047189.70340112 6355591.99032158 6003155.5796842 6236809.42944976 6549017.15295703 5691168.94513392 Histamine 14972.2903581238 7875.38597229005 1413.04188919061 3903.39618667608 5962.7929786682 14610.8799297486 9313.4033752441 20594.0847737122 24259.1792509463 16331.0825268549 13369.5006906127 18786.0715002439 2920.31863662724 16214.2601156617 7874.0329156494 15276.2842517088 13452.1470936584 9458.90488082882 Histidine 1190482.92057892 947942.971432678 840259.970496154 780395.699642641 632102.148033448 1064099.53964917 1195854.28078143 1324186.42609735 1442690.92948907 1227155.94816711 1373906.141612 1455795.68740726 1060272.27005396 1316259.92246197 1322578.48381561 1388742.44042609 1346615.01650632 1291787.68851526 135.036@6.09, provisional ID = Homocysteine 103115.711414795 82243.465734253 76984.0890179443 97189.7974444141 74379.0379699707 136966.052958994 108548.73225769 114464.338407818 120604.719618807 96342.9544972936 114062.001926754 124667.540241331 91068.9031890869 118248.142243933 111881.746591889 110571.073701332 118468.92321109 104545.091302618 Hypotaurine 80627.5173849486 59263.7094253538 62919.0538500974 57665.9694287112 44482.8381152345 114112.574341431 95462.8768481449 132243.175421509 146686.741282349 102103.473604126 122457.083757325 134177.45664917 84336.0565142821 119726.358850708 132580.362286377 124599.625686035 114971.141118774 113037.520924682 Inosine 7079786.08108269 5163321.43956779 5235644.17741008 5726618.83809758 5068049.30403403 9389049.47866597 6335810.48722815 8159745.55374316 9032800.14625915 6770782.95114779 8883794.58285843 8729563.78372498 6594349.20364885 7700376.75733518 7763147.98946968 7886754.94960477 8854889.18171516 6775259.04726973 L-Monomethylarginine 27789.7722290038 26649.2793872834 25741.8828093719 31120.3419885254 21570.5769554139 27316.8297442628 31286.2540773011 29680.1500994873 29638.2534723663 28164.1107413483 31836.233407669 29000.789543762 28986.6044156189 31461.6734546661 31275.6816564179 29874.6605329894 26710.5260388949 31189.5795857238 CAR 18:3 127683.320157166 123215.305416565 113908.766550293 100658.156383057 73557.6031958006 139640.049194336 135 135 135 135 135 135 135 135 135 135 135 135 CAR 18:2 1510388.96840759 1461698.90195373 1366502.73791504 1297959.81705444 944453.99577637 1706743.10417236 87309.691434326 140245.859708862 105112.473848266 69430.8012805175 96085.9110903926 88646.1719195555 79968.325708618 95932.5782000733 72910.1542047116 70982.4020214842 90470.7557147216 60932.4798432618 Lysine 551331.18861847 470668.697783814 437554.748023498 540804.863579986 465280.756559143 640750.447121948 562827.305866149 553799.236904052 633729.458994139 584361.835110354 606184.517410157 604016.699909119 464206.881932191 582308.051179075 606043.924685484 610597.855658753 602371.63414978 591177.459145569 LPC 16:0 33648267.6811966 35175426.458019 30875438.5945401 30172167.5621237 21949217.7378211 39004449.5148268 2903578.72759143 3582347.00224817 2792286.55453516 2166133.09842163 2637357.51057434 2770961.14569153 2352241.57269983 2473430.03663391 2075508.98578162 1932383.85748657 2361262.36004846 2023562.11877704 LPC 16:1 692129.593699952 736641.725850709 678710.133916382 600910.074611206 476673.812722168 688989.824163817 42794.0897940063 70684.6476599121 51202.5731768799 26625.1748855593 45200.3838726808 32568.3292187503 33664.5637198488 39494.8146789551 31691.1668780517 12294.7728167726 35692.8876239014 24163.9420791626 LPC 18:0 39053850.005168 39060097.1176441 36222756.7766022 33395113.0359056 25810607.9574347 42673207.8217318 4706274.73548097 5546351.73490051 4990258.99430968 3136440.54766815 3786207.76784545 4216373.40420899 3667931.64049622 4047651.85784729 3418503.31813721 2832367.06956421 3308404.4685658 3030429.27367382 LPC 18:1 18148166.3924879 18227051.3966312 17286505.8596197 16017348.9668076 12736566.7028693 19284182.4731214 1668401.39076007 2247343.48014526 1788596.12746612 1230588.969151 1591293.00196258 1536530.16154785 1352949.01989947 1508764.77680786 1344599.67121826 1125940.12401337 1429994.92366095 1158489.6248371 LPC 18:2 23981564.3809022 26661532.5575581 24681599.9665975 21016926.6499776 16681733.2516772 24434853.5366496 2057235.76887024 2938575.90056121 2125077.29352527 1415809.16310364 1807232.79422089 1756725.66089935 1645776.10540307 1913463.60790985 1682161.73055023 1244541.7281792 1590198.00688648 1398646.70968604 LPC 18:3 403026.021783142 455389.57328888 413701.693681945 332990.679326782 259121.162773133 392959.073202209 16374.8373352051 28473.6568777465 21371.4317456055 38968.0116229247 13305.1704074096 10163.7717294311 7852.50552764884 17750.5865093994 6669.2644012451 11048.0269558714 12762.1833740233 70837.8963720094 LPE 18:2 199076.511652832 209811.485911255 162044.191728515 159813.104989502 142383.425892822 202371.63539917 5377.84525756836 135 135 21980.7380603028 2756.13300354 3281.80592773425 12805.5286791992 4841.73218322754 135 11844.9905230714 135 77911.188695678 CAR 4:1;O2 598579.308062134 482467.302731934 432402.735721801 484345.604499206 431395.5698172 848473.969485474 636772.998372192 725402.39770752 794824.621750791 642562.385838621 767563.804577698 785707.788198608 531267.558978271 734475.280642088 733928.302648925 715019.169266052 757685.115097655 677587.889905397 N1-Acetylspermidine 59852.9342889785 41272.2166360854 47982.0759925845 55162.9006331638 40506.5679007725 70209.9489296116 55085.0118119434 57402.0356677247 69317.824261627 58108.9476999665 66096.9213905332 65807.217703857 41223.4675671693 65780.5818243407 61170.5968463899 60987.7960531614 63045.6450321956 58649.7244763181 Nicotinamide 157272503.913171 126380363.443642 130397286.668273 140847332.751462 122273327.156303 207675119.903265 158857381.561079 189687810.16427 195215731.741438 162129725.050849 194162886.194371 192905074.581971 160961577.863893 181261258.101454 176681957.591407 189513630.597573 189962783.680406 147109291.136424 325.2973@1.13, provisional ID = NAE 18:1 401499.321258179 376533.994160156 267016.62979303 300310.54405603 239670.843496338 241298.564907959 185480.020103516 245583.643793457 236762.453959228 225144.939876709 338436.738663574 133617.035259033 176020.860510132 192176.382961792 153385.050657471 211746.506591064 200736.751506836 208256.345479309 CAR 18:1 5321788.23812842 5180764.08528529 4627111.94588868 4459218.99757781 3368046.29952331 5513063.16248132 392175.988763734 527368.363214784 438912.164979858 311503.000873597 371820.540086669 366936.47315033 318078.609136353 372067.561487732 316885.210057801 356603.287898255 357753.855108765 299445.879558898 Ornithine 200742.418308899 191936.028122406 193848.785042786 204304.112408508 192899.084434723 265474.821530792 227894.384479217 241636.388676483 259501.390277039 225858.533480407 259508.502415344 255556.7098573 187463.319972747 248197.752218842 250054.40171759 251710.476975006 235536.242795684 241689.927038543 299.2813@1.17, provisional ID = SPB 18:1;O2 470513.665451415 366011.024304077 396996.485428589 374678.982876465 349940.083727051 540161.574177491 141397.217896606 156016.240289795 124179.506771606 162697.357255737 94174.4692103271 150405.554344727 153730.93273999 137414.823551941 162314.569958984 113277.602245239 129577.134986084 70996.7289227904 CAR 16:0 1408599.57020923 1406342.08469697 1327309.93437103 1223392.30876221 866093.537998656 1912820.62916688 143132.55855896 209100.145944214 177510.380169372 133975.519003296 157294.673686402 144513.83396637 141661.689066772 150332.836750794 128187.324669189 133395.27651001 158136.630543213 109592.418917053 PC 34:2 59195276.0303434 71207158.5699787 66093820.2591335 60153099.6425757 46977895.5302549 78405077.7864344 14205004.8407473 13925704.1481863 13735972.7769376 8883657.24587829 10683555.1695231 12554418.5819024 9864996.17639888 11725002.6998783 9822471.55132569 8518938.95072364 9596055.21753286 7633083.58936779 PC 36:2 14417371.6129326 16794741.6631807 15969569.4455637 14428727.0120069 11778163.065662 18021753.5845424 3835259.94906652 3676255.46976874 3624501.69288373 2332112.65479578 2924713.32438037 3317435.82745154 2625219.15453369 3119796.16106164 2614147.22878271 2262918.89037696 2632261.87769977 1987882.24286841 PC 36:4 37935281.3213607 44205997.0605058 42928378.6975158 37503145.1140921 30255761.383343 48932899.8632798 9680286.35053043 9600500.30263682 9713710.2074852 5921118.98637559 7533856.78157031 8819129.78759927 7100259.76856016 8227623.85052428 6858328.824812 6002758.79731278 6692581.98128214 5220956.42041822 Phosphocholine 2187479.14615195 1817265.36484784 1564121.00714374 1891162.46240765 1565102.0842605 2985220.46920013 2344759.13708154 2594974.40805133 2842766.601526 2475080.20889105 2791889.14919354 2874488.8419874 1917779.91587396 2619700.66873902 2619235.00765357 2530663.23543463 2651131.38698157 2453902.1414519 Pipecolinic Acid 223181.323743896 172915.978036499 199347.774045105 175961.619655579 146340.824538208 310065.937518677 197428.317463746 292041.87203772 253235.553024536 215656.837887207 314938.563286987 271563.422833496 225478.796307252 266873.124287964 258492.763720702 282482.636863525 256910.88695984 238891.58948645 Proline Betaine 12065171.4970962 10001295.5133052 8814151.27282415 10804082.4163164 9357935.03288643 16455542.5873839 12600274.7171804 13825234.4047208 16170709.1755157 13450062.7004606 15655910.4483461 15938219.5530053 10508081.9715981 14614772.7373442 14956214.4316979 14402087.9594663 15663437.4835431 13803111.2830804 CAR 3:0 5546033.34900293 4635185.1560608 4031737.52724194 4828858.5463485 4237130.06144348 7636771.03304449 5958390.11650763 6411184.55536164 7329442.587679 6204295.05636821 7233719.73544164 7286867.86421912 4786253.18879968 6651818.1326812 6788516.99979766 6517819.00278327 6912922.57057013 6509789.03762684 S-Adenosylmethionine 48172.5663709411 37632.9130395813 38080.5679777374 37601.842611923 23081.4485167696 94329.5482523346 40251.8623483274 73331.1363556366 90862.1121855317 46605.9811334991 83787.9564853667 80195.8676355438 30649.7579331206 72456.3180624389 70551.6278238679 73775.0739452363 70113.8644499818 67550.1399488984 Succinylaminoimidazole carboxamide riboside 14963.2250724793 7295.29552825931 8639.48424182135 6807.37601013179 5256.31525604248 15356.3688671112 14128.6073855592 20807.3723815918 23025.3185043335 15835.8961447145 19036.0559838869 15345.4271664429 11351.4271895753 18739.673336792 14031.3442028808 19009.8128570557 21599.9190087891 9990.11294311519 SM d34:0 63034.1318600466 82839.8401083374 76642.2982843019 62134.8031756592 40396.7597155761 118284.555118408 135 135 135 135 135 135 135 135 135 135 135 135 SM d34:1 1229336.30786487 1580430.36872437 1536608.93458411 1314296.34427735 872568.73545349 2002464.19533105 187427.226573486 208264.095420227 173566.229269714 104809.785215759 131376.758796387 157234.594640503 115415.096109619 141937.861278381 103471.265574341 82588.9226116941 102791.726072998 56928.3179135132 SM 36:2;O2 26074.4997329713 41920.8718896484 29345.1393087769 23009.318197632 14321.7577960205 56478.4018649294 135 135 135 135 135 135 135 135 135 135 135 135 Glycerophosphocholine 31913571.342519 26555598.3892373 23736466.1076656 27903548.2024607 23540108.739372 44516013.8725204 33639395.0734334 39038891.2901735 42670177.6955104 35446543.205024 41065238.8905882 41809511.1546046 29453157.7343727 38155677.2788396 37956856.8826513 36610012.3229995 38684138.4683215 36799194.7208734 SM 36:1;O2 263948.794249512 334520.901821289 316208.168497315 285809.430709229 166803.795748291 450891.010701904 16543.8651010132 24252.4387438965 18926.0562832641 6068.84382080064 4768.65573608397 11794.3873193361 7405.02432678225 8061.73257232665 3949.14053833013 4472.28205444342 6793.38941711428 135 CAR 18:0 1300655.94462854 1302388.9875113 1153243.52484009 1065377.85373321 830417.774876708 1605255.30247651 145897.675795593 181971.570576539 156472.547882386 108311.990840149 147174.031401367 151026.528342591 128086.814780579 142002.897167053 126749.969619446 185311.705917664 195294.121897278 204939.197733703 Succinoadenosine 87229.8086802367 57468.3579947206 63889.6877738952 74844.4285134885 65286.2246255492 93575.1816474914 134731.048724365 159895.13058197 153391.900332947 139254.256774781 165133.150253906 153490.736104737 129389.942208496 147087.730209168 137654.242061157 148656.172846679 147785.975384704 110920.654436463 Dimethylarginine 74464.6675343015 66882.6892560581 64588.2320085906 68931.9263745117 71515.3547425383 90547.5818612672 71610.468579178 82260.9646650696 87389.57923378 73355.3321622466 93689.9474259185 79206.5881072236 69822.8484031067 84254.2322373199 79339.9847353361 73431.9811914059 76697.1139155881 80362.7842446903 CAR 14:1 58027.9598150635 55185.4359381103 55029.0372259521 46189.2521368409 31029.5003729248 66216.4628179929 135 135 135 135 135 2058.37909179679 135 135 1411.19736816408 2458.54583557132 1024.00860382076 135 Thiamine 212363.029675292 168724.788388024 148663.830177766 178497.5518425 140871.533338395 348061.889540971 219986.60066658 267740.583027873 295009.586531677 225769.449257814 289030.478360293 287661.139453568 170670.211905168 256921.922616039 257992.661559444 254614.115534822 264840.213950196 268997.90685249 Trimethyllysine 65740.0118421173 58737.8042474366 58678.3410245513 60719.6511335755 54802.4004506682 97109.8568242649 76899.4574331664 80037.7598188782 87779.3426281741 75451.0383295441 83664.3622698972 86303.2396733096 67423.6008177188 79815.1780185318 77963.8123674774 82394.5041952511 85492.8916826623 79614.1187297059 Tyramine 292294.115795288 310108.296693725 290979.162238159 288313.134567871 326628.139118408 252055.348625122 303651.384856201 269169.931715088 265633.284962891 310034.003997558 280474.837225342 277171.545174805 279535.734985352 282149.786137695 296735.859496582 290254.820278321 236701.930289306 301955.062091065 Urea 3066954.76644922 2766733.93093457 3938197.02890625 4098599.91259179 2696176.8306084 4622361.45355078 4279307.75876758 5508676.99169043 5483505.71155762 4512915.9685586 4932660.53247071 5149345.33813867 3928893.94514063 3985479.5340957 4200123.94204004 4246661.5393125 5084553.00704687 3922868.15326172 CAR 5:0 2577471.6386106 2054080.18618506 2013975.97580969 2239463.26231958 1870845.64448224 3705956.66878089 2714163.10775129 3272304.95987183 3516492.13948755 2755811.92764356 3483991.50933667 3306686.25759302 2445251.53606635 3229201.43660431 3044546.54558984 3197416.37247076 3380991.29987146 33737.0743990479 Xanthine 284736.99430896 212858.532877197 191394.576355469 235989.290505249 208596.041479492 311704.660008545 250291.878775024 276597.845591492 309513.774103638 260210.705586303 292552.024044189 308917.475380737 228399.912812256 272738.26688855 295021.482383606 270718.601651062 296656.055119629 256370.801306152 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Pubchem ID Formula Mass (measured) RT Mass (actual) InChiKeys 1-Methyladenosine 27476 C11H15N5O4 281.1119 9.19 281.1124 GFYLSDSUCHVORB-IOSLPCCCSA-N 1-Methylnicotinamide 457 C7H8N2O 136.0639 26.57 136.0637 LDHMAVIPBRSVRG-UHFFFAOYSA-O 135.036@6.09, provisional ID = Homocysteine C4H9NO2S 135.0323 3.6 135.0354 299.2813@1.17, provisional ID = SPB 18:1;O2 C18H37NO2 299.2793 1.55 299.2824 3-Methylhistamine 69520 C6H11N3 125.0957 26.59 125.0953 CPAGZVLINCPJEH-UHFFFAOYSA-N 3-Methylhistidine 64969 C7H11N3O2 169.0855 12.13 169.0851 JDHILDINMRGULE-LURJTMIESA-N 323.2824@1.13, provisional ID = NAE 18:2 C20H37NO2 323.2785 1.53 323.2824 325.2973@1.13, provisional ID = NAE 18:1 C20H39NO2 325.2934 1.52 325.2981 367.2674@7.75, provisional ID = CAR 14:2 C21H37NO4 367.2734 10.42 367.2723 4-Aminobutyric Acid C4H9NO2 103.0636 9.35 103.0633 4-Guanidinobutyric Acid 500 C5H11N3O2 145.0852 11.7 145.0851 TUHVEAJXIMEOSA-UHFFFAOYSA-N 4-Imidazoleacetic Acid 96215 C5H6N2O2 126.0433 9.71 126.0429 PRJKNHOMHKJCEJ-UHFFFAOYSA-N 441.355@7.97, provisional ID = CAR 18:1-0H C25H47NO5 441.3459 10.48 441.3454 5-Aminopentanoic Acid C5H11NO2 117.0793 13.92 117.079 5'-Methylthioadenosine C11H15N5O3S 297.0899 2.49 297.0896 526.4679@1.14, provisional ID = DG(29:0) C32H62O5 526.4497 0.75 526.4597 538.3788@7.87, provisional ID = PC O-20:0 C26H55N2O7P 538.3824 10.58 538.3747 Adenine 190 C5H5N5 135.0548 4.51 135.0545 GFFGJBXGBJISGV-UHFFFAOYSA-N Adenosine 60961 C10H13N5O4 267.0973 3.45 267.0968 OIRDTQYFTABQOQ-KQYNXXCUSA-N Alanine 5950 C3H7NO2 89.0476 9.66 89.0477 QNAYBMKLOCPYGJ-REOHCLBHSA-N Arginine 6322 C6H14N4O2 174.1164 26.58 174.1117 ODKSFYDXXFIFQN-BYPYZUCNSA-N Betaine 247 C5H11NO2 117.0798 11.29 117.079 KWIUHFFTVRNATP-UHFFFAOYSA-N Bilirubin 5280352 C33H36N4O6 584.2741 1 584.2635 BPYKTIZUTYGOLE-IFADSCNNSA-N CAR 12:0 C19H37NO4 343.2732 11.02 343.2723 CAR 14:1 C21H39NO4 369.2892 10.34 369.2879 CAR 16:0 C23H45NO4 399.3353 10.19 399.3349 CAR 16:0;O C23H45NO5 415.33 11.43 415.3298 CAR 16:1 C23H43NO4 397.3198 10.23 397.3192 CAR 16:1;O C23H43NO5 413.3152 11.13 413.3141 CAR 18:0 C25H49NO4 427.3663 10.05 427.3662 CAR 18:1 C25H47NO4 425.3509 10.09 425.3505 CAR 18:2 C25H45NO4 423.3355 10.14 423.3349 CAR 18:3 C25H43NO4 421.3207 10.15 421.3192 CAR 2:0 C9H17NO4 203.1166 14.2 203.1158 CAR 3:0 C10H19NO4 217.132 13.48 217.1314 CAR 4:0 C11H21NO4 231.1477 12.82 231.1471 CAR 4:1;O2 C11H19NO6 261.122 13.43 261.1212 CAR 5:0 C12H23NO4 245.163 12.58 245.1627 CAR 6:0 C13H25NO4 259.1787 11.67 259.1784 Carnitine 10917 C7H15NO3 161.1061 14.88 161.1052 PHIQHXFUZVPYII-ZCFIWIBFSA-N Choline 305 C5H13NO 103.1 26.57 103.0997 OEYIOHPDSNJKLS-UHFFFAOYSA-N Citrulline 9750 C6H13N3O3 175.0961 12.13 175.0957 RHGKLRLOHDJJDR-BYPYZUCNSA-N Creatinine 588 C4H7N3O 113.0593 8.83 113.0589 DDRJAANPRJIHGJ-UHFFFAOYSA-N Cytidine 6175 C9H13N3O5 243.0871 5.56 243.0855 UHDGCWIWMRVCDJ-XVFCMESISA-N Deoxycarnitine 134 C7H15NO2 145.1104 18.1 145.1103 JHPNVNIEXXLNTR-UHFFFAOYSA-O Dimethylarginine 123831 C8H18N4O2 202.1429 26.61 202.143 YDGMGEXADBMOMJ-LURJTMIESA-N Glycerophosphocholine C8H20NO6P 257.1037 15.11 257.1028 Guanidinosuccinic Acid 97856 C5H9N3O4 175.0596 9.66 175.0593 VVHOUVWJCQOYGG-UHFFFAOYSA-N Guanine 764 C5H5N5O 151.0504 3.91 151.0494 UYTPUPDQBNUYGX-UHFFFAOYSA-N Guanosine 6802 C10H13N5O5 283.092 3.87 283.0917 NYHBQMYGNKIUIF-UUOKFMHZSA-N Histamine 774 C5H9N3 111.081 26.71 111.0796 NTYJJOPFIAHURM-UHFFFAOYSA-N Histidine 6274 C6H9N3O2 155.0699 11.65 155.0695 HNDVDQJCIGZPNO-YFKPBYRVSA-N Hypotaurine 107812 C2H7NO2S 109.0198 10.09 109.0197 VVIUBCNYACGLLV-UHFFFAOYSA-N Inosine 6021 C10H12N4O5 268.0818 3.31 268.0808 UGQMRVRMYYASKQ-KQYNXXCUSA-N L-Monomethylarginine C7H16N4O2 188.1282 26.59 188.1273 LPC 16:0 C24H50NO7P 495.3335 10.31 495.3325 LPC 16:1 C24H48NO7P 493.3171 10.41 493.3168 LPC 18:0 C26H54NO7P 523.3649 10.11 523.3638 LPC 18:1 C26H52NO7P 521.3488 10.2 521.3481 LPC 18:2 C26H50NO7P 519.3332 9.99 519.3325 LPC 18:3 C26H48NO7P 517.3172 9.99 517.3168 LPE 18:2 C23H44NO7P 477.286 7.09 477.2855 Lysine 5962 C6H14N2O2 146.1068 26.58 146.1055 KDXKERNSBIXSRK-YFKPBYRVSA-N N1-Acetylspermidine 496 C9H21N3O 187.1685 26.59 187.1685 MQTAVJHICJWXBR-UHFFFAOYSA-N Nicotinamide 936 C6H6N2O 122.0487 2.43 122.048 DFPAKSUCGFBDDF-UHFFFAOYSA-N Ornithine 6262 C5H12N2O2 132.09 26.25 132.0899 AHLPHDHHMVZTML-BYPYZUCNSA-N PC 34:2 C42H80NO8P 757.5641 8.44 757.5622 PC 36:2 C44H84NO8P 785.5944 8.34 785.5935 PC 36:4 C44H80NO8P 781.564 8.25 781.5622 PC O-36:4 70698779 C44H82NO7P 767.5727 9.03 767.5829 XWYIIYJIVUGRQE-AMUBJDAWSA-N Phosphocholine 1014 C5H14NO4P 183.0662 19.69 183.066 YHHSONZFOIEMCP-UHFFFAOYSA-O Pipecolinic Acid 736316 C6H11NO2 129.0793 9.88 129.079 HXEACLLIILLPRG-RXMQYKEDSA-N Proline Betaine 115244 C7H13NO2 143.0948 11.73 143.0946 CMUNUTVVOOHQPW-LURJTMIESA-N S-Adenosylmethionine C15H22N6O5S 398.1369 22.02 398.1372 SM 36:1;O2 C41H83N2O6P 730.5989 9.33 730.5989 SM 36:2;O2 C41H81N2O6P 728.5832 9.32 728.5832 SM d34:0 C39H82N2O6P 705.5861 9.32 705.591 SM d34:1 C39H80N2O6P 703.5716 9.52 703.5754 ST 21:4;O5 C21H28O5 360.196 1.01 360.1937 Succinoadenosine 20849086 C14H17N5O8 383.108 8.68 383.1077 VKGZCEJTCKHMRL-VWJPMABRSA-N Succinylaminoimidazole carboxamide riboside C13H18N4O9 374.1078 8.83 374.1074 Thiamine C12H16N4OS 264.1046 26.55 264.1045 Trimethyllysine 440121 C9H20N2O2 188.1525 26.6 188.1525 MXNRLFUSFKVQSK-QMMMGPOBSA-O Tyramine 5610 C8H11NO 137.0844 11.37 137.0841 DZGWFCGJZKJUFP-UHFFFAOYSA-N Urea 1176 CH4N2O 60.0318 2.81 60.0324 XSQUKJJJFZCRTK-UHFFFAOYSA-N Xanthine 1188 C5H4N4O2 152.0345 2.43 152.0334 LRFVTYWOQMYALW-UHFFFAOYSA-N METABOLITES_END #END