#METABOLOMICS WORKBENCH jad2033_20230605_165439 DATATRACK_ID:4066 STUDY_ID:ST002744 ANALYSIS_ID:AN004449 PROJECT_ID:PR001509 VERSION 1 CREATED_ON June 26, 2023, 11:04 am #PROJECT PR:PROJECT_TITLE Biomolecular condensates create phospholipid-enriched microenvironments PR:PROJECT_TYPE Metabolomes of in vitro synthesized 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 srj2003@med.cornell.edu PR:PHONE 6466590174 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. Jaffrey #STUDY ST:STUDY_TITLE Biomolecular condensates create phospholipid-enriched microenvironments (Part 4) 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. Here, we confirmed the presence of lipids in ST:STUDY_SUMMARY MED1 samples and condensates using reversed-phase LC-MS. ST:INSTITUTE Cornell University ST:LAST_NAME Dumelie ST:FIRST_NAME Jason ST:ADDRESS 1300 York Ave, LC-524, New York City, NY ST:EMAIL srj2003@med.cornell.edu ST:PHONE 6466590174 #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 Aqueous Sample 4 fraction:aqueous | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; RAW_FILE_NAME=51_T_No650.mzdata.xml, p_51_T_No650.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Aqueous Sample 5 fraction:aqueous | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; RAW_FILE_NAME=52_T.mzdata.xml, p_52_T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Aqueous Sample 6 fraction:aqueous | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; RAW_FILE_NAME=53_T.mzdata.xml, p_53_T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Aqueous Sample 7 fraction:aqueous | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; RAW_FILE_NAME=61_T.mzdata.xml, p_61_T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Aqueous Sample 8 fraction:aqueous | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; RAW_FILE_NAME=62_T.mzdata.xml, p_62_T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Aqueous Sample 9 fraction:aqueous | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; RAW_FILE_NAME=63_T.mzdata.xml, p_63_T.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Condensate Sample 4 fraction:condensate | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; RAW_FILE_NAME=51_B_No650.mzdata.xml, p_51_B_No650.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Condensate Sample 5 fraction:condensate | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; RAW_FILE_NAME=52_B.mzdata.xml, p_52_B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Condensate Sample 6 fraction:condensate | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; RAW_FILE_NAME=53_B.mzdata.xml, p_53_B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Condensate Sample 7 fraction:condensate | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; RAW_FILE_NAME=61_B.mzdata.xml, p_61_B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Condensate Sample 8 fraction:condensate | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; RAW_FILE_NAME=62_B.mzdata.xml, p_62_B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Condensate Sample 9 fraction:condensate | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; RAW_FILE_NAME=63_B.mzdata.xml, p_63_B.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 4 fraction:input | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; RAW_FILE_NAME=51_I_No650.mzdata.xml, p_51_I_No650.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 5 fraction:input | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; RAW_FILE_NAME=52_I.mzdata.xml, p_52_I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 6 fraction:input | Extraction from condensate:No heat step Protein=MED1; RNA=150 nM; timing of metabolite extract addition=standard; RAW_FILE_NAME=53_I.mzdata.xml, p_53_I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 7 fraction:input | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; RAW_FILE_NAME=61_I.mzdata.xml, p_61_I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 8 fraction:input | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; RAW_FILE_NAME=62_I.mzdata.xml, p_62_I.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Input Sample 9 fraction:input | Extraction from condensate:standard Protein=MED1; RNA=150 nM; timing of metabolite extract addition=Metabolites added after 10 min incubation; RAW_FILE_NAME=63_I.mzdata.xml, p_63_I.mzdata.xml #COLLECTION CO:COLLECTION_SUMMARY Mouse metabolites were collected from the liver of female mice using methanol CO:COLLECTION_SUMMARY extraction. After euthanizing a mouse, the liver was immediately frozen in CO:COLLECTION_SUMMARY liquid nitrogen. We then used cold 80% methanol to extract metabolites. First, 1 CO:COLLECTION_SUMMARY ml of 80% methanol was added to the liver and incubated for 10 min at -20oC. CO:COLLECTION_SUMMARY Glass beads were added to the liver and then the liver was lysed by bead-beating CO:COLLECTION_SUMMARY for 45 s using a Tissuelyser cell disrupter (Qiagen). The lysate was incubated CO:COLLECTION_SUMMARY for 10 min at -20oC and centrifuged (13200 rpm, 5 min) to separate metabolites CO:COLLECTION_SUMMARY from macromolecules. The supernatant was collected and 200 µl of 80% methanol CO:COLLECTION_SUMMARY was added to the pellet. The incubation, shaking and centrifugation steps were CO:COLLECTION_SUMMARY repeated twice to extract more metabolites from the pellet. The three CO:COLLECTION_SUMMARY supernatants were combined and centrifuged (14000 rpm, 10 min) to separate any CO:COLLECTION_SUMMARY remaining macromolecules from the metabolites. The combined supernatants were CO:COLLECTION_SUMMARY dried using a SpeedVac Concentrator (Savant, SPD131DDA) at 25oC and the dried CO:COLLECTION_SUMMARY metabolite samples were stored at -80oC. The amount of protein in the pellet was CO:COLLECTION_SUMMARY measured using the Quick Start Bradford assay to calculate the metabolites’ CO:COLLECTION_SUMMARY protein equivalent mass. Mouse metabolites were initially re-suspended in CO:COLLECTION_SUMMARY condensate buffer (50 mM NH4HCO3 pH 7.5, 50 mM NaCl, 1 mM DTT) to a protein CO:COLLECTION_SUMMARY equivalent concentration of 938 g/l. The chosen final concentration of CO:COLLECTION_SUMMARY metabolites is slightly lower than the 200-300 g/l protein concentration CO:COLLECTION_SUMMARY observed in cells. Metabolites that were not fully soluble in condensate buffer CO:COLLECTION_SUMMARY were removed by centrifugation (2x5 min, 16,000 g each), in which only the CO:COLLECTION_SUMMARY supernatant was retained. Purified mCherry tagged MED1 low-complexity domain CO:COLLECTION_SUMMARY (37.5 μM) was centrifuged (1 min, 1,000 g) to disrupt any existing condensates CO:COLLECTION_SUMMARY and to remove any precipitated proteins. The MED1 (final concentration, 30 μM) CO:COLLECTION_SUMMARY was then combined with metabolites (final concentration, 150 g/l protein CO:COLLECTION_SUMMARY equivalent) and then phage lambda RNA (final concentration, 0.15 μM) in a total CO:COLLECTION_SUMMARY volume of 300 µl. An input sample (10 µl) was saved and then the sample was CO:COLLECTION_SUMMARY allowed to incubate for 10 min at 25oC. Condensates were then separated from the CO:COLLECTION_SUMMARY aqueous environment by centrifugation (10 min, 12,500 g, 25oC). The aqueous CO:COLLECTION_SUMMARY phase was removed from the condensate phase and then equal volumes (usually ~ 2 CO:COLLECTION_SUMMARY µl) of the aqueous fraction, condensate fraction and input sample were CO:COLLECTION_SUMMARY processed for metabolomics using identical approaches as described below. First CO:COLLECTION_SUMMARY the samples were diluted in ammonium bicarbonate buffer (50 mM NH4HCO3 pH 7.5) CO:COLLECTION_SUMMARY and briefly heated (2 min, 65oC) to disrupt condensates before being added CO:COLLECTION_SUMMARY immediately to 4x volume of ice-cold 100% methanol to precipitate protein and CO:COLLECTION_SUMMARY RNA. This heating step was excluded for some samples where noted. Protein and CO:COLLECTION_SUMMARY RNA were separated from metabolites by vortexing the samples (2 min), followed CO:COLLECTION_SUMMARY by incubation at -25oC (10 min) and then centrifugation (5 min, 13,000 rpm). The CO:COLLECTION_SUMMARY supernatant was saved and the process was repeated on the pellet two more times CO:COLLECTION_SUMMARY after adding 200 µl of 80% methanol each time to the pellet. The three CO:COLLECTION_SUMMARY supernatants were combined and centrifuged (10 min, 14000 rpm) to remove any CO:COLLECTION_SUMMARY additional macromolecules. The final supernatant was collected and dried using a CO:COLLECTION_SUMMARY SpeedVac Concentrator run at 25oC. Notably, in a subset of experiments, CO:COLLECTION_SUMMARY metabolites were added to MED1 condensates after the 10 min incubation rather CO:COLLECTION_SUMMARY than prior to the incubation. CO:SAMPLE_TYPE Liver CO:COLLECTION_METHOD 80% methanol CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Mouse liver metabolites were combined with the condensate-forming low-complexity TR:TREATMENT_SUMMARY domain of MED1. Condensates were stimulated with 150 nM RNA and then incubated TR:TREATMENT_SUMMARY for 10 min. In a subset of samples, RNA addition occurred 10 min before TR:TREATMENT_SUMMARY metabolite addition. Next, condensates were centrifuged to the bottom of a 600 TR:TREATMENT_SUMMARY ul tube. Equal fractions from the input sample, aqueous phase and condensate TR:TREATMENT_SUMMARY phases were collected separately. Metabolites were extracted from each fraction TR:TREATMENT_SUMMARY using 80% methanol in steps that involved disrupting condensates with heat. In a TR:TREATMENT_SUMMARY subset of samples, this heat step was omitted. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Dried-down extracts were reconstituted in 150 µl 70% acetonitrile, at a SP:SAMPLEPREP_SUMMARY relative protein concentration of ~ 2 µg/µl. These were stored at -20C until SP:SAMPLEPREP_SUMMARY used. In these LC-MS experiments, 4 µl of reconstituted extract was injected SP:SAMPLEPREP_SUMMARY for LC/MS-based targeted metabolite profiling for each sample. SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:EXTRACT_STORAGE -20℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY This reversed phase platform was adapted with minor modifications from a CH:CHROMATOGRAPHY_SUMMARY previously reported Agilent application note (A Comprehensive, Curated, CH:CHROMATOGRAPHY_SUMMARY High-Throughput Method for the Detailed Analysis of the Plasma Lipidome, CH:CHROMATOGRAPHY_SUMMARY https://www.agilent.com/cs/library/applications/an-plasma-lipidomics-6495-lc-ms-ms-5994-3747en-agilent.pdf) CH:CHROMATOGRAPHY_SUMMARY and showed similar lipid retention times for fatty acids and other lipids, as CH:CHROMATOGRAPHY_SUMMARY reported. This platform was comprised of an Agilent Model 1290 Infinity II CH:CHROMATOGRAPHY_SUMMARY liquid chromatography system coupled to an Agilent 6550 iFunnel time-of-flight CH:CHROMATOGRAPHY_SUMMARY MS analyzer. An Agilent ZORBAX Eclipse Plus C18, 100 × 2.1 mm, 1.8 μm reversed CH:CHROMATOGRAPHY_SUMMARY phase column was used for the separation. Mobile phases consisted of (A) 10 mM CH:CHROMATOGRAPHY_SUMMARY ammonium formate with 5 μM Agilent deactivator additive in 5:3:2 CH:CHROMATOGRAPHY_SUMMARY water:acetonitrile:2-propanol and (B) 10 mM ammonium formate in 1:9:90 CH:CHROMATOGRAPHY_SUMMARY water:acetonitrile:2-propanol. Column temperature was set at 55°C and CH:CHROMATOGRAPHY_SUMMARY autosampler temperature was at 20°C. The flow rate was 0.4 mL/min. The CH:CHROMATOGRAPHY_SUMMARY following gradient was applied: 0 min, 15% B; 0-2.5 min, to 50% B; 2.5-2.6 min, CH:CHROMATOGRAPHY_SUMMARY to 57%, 2.6-9 min, to 70% B; 9-9.1 min, to 93% B; 9.1-11.1 min, to 96%; 11.1- CH:CHROMATOGRAPHY_SUMMARY 15min, 100% B; 15-20 min, 15% B. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um) CH:SOLVENT_A 50% water/30% acetonitrile/20% isopropanol;10 mM ammonium formate with 5 µM CH:SOLVENT_A Agilent deactivator additive CH:SOLVENT_B 1% water/9% acetonitrile/90% isopropanol;10 mM ammonium formate CH:FLOW_GRADIENT 0 min, 15% B; 0-2.5 min, to 50% B; 2.5-2.6 min, to 57%, 2.6-9 min, to 70% B; CH:FLOW_GRADIENT 9-9.1 min, to 93% B; 9.1-11.1 min, to 96%; 11.1- 15min, 100% B; 15-20 min, 15% CH:FLOW_GRADIENT B. CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 55 #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 Raw data were analyzed using Mass Hunter Qualitative analysis (10.0), MassHunter MS:MS_COMMENTS Profinder 8.0 and MassProfiler Professional (MPP) 15.1 software (Agilent MS:MS_COMMENTS technologies). Lipid identification was confirmed in the original samples by MS:MS_COMMENTS comparison with the retention times and masses in a lipidomics PCDL of 665 MS:MS_COMMENTS metabolites developed as recommended by the manufacturer (Agilent). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Ion abundance MS_METABOLITE_DATA_START Samples MED1 Condensate Sample 4 MED1 Condensate Sample 5 MED1 Condensate Sample 6 MED1 Input Sample 4 MED1 Input Sample 5 MED1 Input Sample 6 MED1 Aqueous Sample 4 MED1 Aqueous Sample 5 MED1 Aqueous Sample 6 MED1 Condensate Sample 7 MED1 Condensate Sample 8 MED1 Condensate Sample 9 MED1 Input Sample 7 MED1 Input Sample 8 MED1 Input Sample 9 MED1 Aqueous Sample 7 MED1 Aqueous Sample 8 MED1 Aqueous Sample 9 Factors fraction:condensate | Extraction from condensate:No heat step fraction:condensate | Extraction from condensate:No heat step fraction:condensate | Extraction from condensate:No heat step fraction:input | Extraction from condensate:No heat step fraction:input | Extraction from condensate:No heat step fraction:input | Extraction from condensate:No heat step fraction:aqueous | Extraction from condensate:No heat step fraction:aqueous | Extraction from condensate:No heat step fraction:aqueous | Extraction from condensate:No heat step fraction:condensate | Extraction from condensate:standard fraction:condensate | Extraction from condensate:standard fraction:condensate | Extraction from condensate:standard fraction:input | Extraction from condensate:standard fraction:input | Extraction from condensate:standard fraction:input | Extraction from condensate:standard fraction:aqueous | Extraction from condensate:standard fraction:aqueous | Extraction from condensate:standard fraction:aqueous | Extraction from condensate:standard 2-Aminoadipic Acid 121912.1953125 106179.140625 98245.015625 199117.359375 194639.453125 198308.890625 193687.546875 205179.796875 199527.78125 109365.453125 112523.09375 122825.328125 195465.359375 201602.671875 203178.9375 189292.46875 184318.984375 172367.75 3-Methylhistidine 11589.638671875 10416.80078125 8058.0966796875 13941.8154296875 17926.3515625 16969.44921875 11710.2353515625 16031.0498046875 14376.5751953125 7666.9658203125 6408.6767578125 10175.072265625 13331.044921875 14843.669921875 17043.958984375 15678.142578125 15028.328125 14173.2158203125 5'-Methylthioadenosine 276312.125 167472.15625 156468.40625 263266.25 243404.9375 222567.1875 215654.296875 228915.4375 208266.671875 230425.53125 188632.40625 279701.59375 236880.984375 280539.25 262903.34375 225384.34375 297985.6875 204884.65625 CAR 2:0 4039289.25 3322169.5 2908060 4409179.5 4902830 5722846.5 3552713.75 5108832.5 5127941 3512486.75 3169439 5668289 4643290.5 5284749.5 5659786.5 5086676 5428804 4724586.5 Arginine 2203.57397460938 1681.63513183594 2165.27709960938 1936.72155761719 2156.07470703125 1859.94067382813 1831.33471679688 1715.42456054688 2057.00439453125 1799.00317382813 6945.09326171875 2624.5400390625 1687.06665039063 2867.837890625 2158.74291992188 3879.37182617188 1829.29833984375 2218.00634765625 Betaine 14606063 13755094 13440170 14498178 15300459 16111033 14823956 15784982 14452072 13969025 13224953 16363876 15007233 15630142 15507315 15527225 15260904 14261244 Bilirubin 9342.271484375 8739.8935546875 9555.30078125 2138.42260742188 3045.69360351563 2598.18286132813 3359.64086914063 2778.02807617188 1482.3203125 6663.59814453125 7132.6083984375 11520.0048828125 2218.0419921875 2077.46923828125 3926.16943359375 3915.19702148438 3343 CAR 4:0 2172189.75 1870725 1641258.85461426 2318475.17993164 2669324.75 3033180.75 2094293.125 2747755.81616211 2712565.27282715 1955022.03295898 1665212.75 3147912.36914063 2438069.75 2919307.25 3046706.28894043 2729618.25 2862686.76306152 2580430 Choline 2035692.875 1602418.375 1253547 2097470.5 2221184.25 2269318.5 1691619.25 2416559 2416067.5 1666111 1467885.875 3027386.5 2173448.75 2570198.5 2631359.75 2599003.75 2498715.75 2430137.75 Dimethylarginine 1549.00646972656 1509.68139648438 1442.85852050781 3021.68090820313 3356.93505859375 3032.017578125 2516.96240234375 3581.3076171875 2868.40454101563 1902.10241699219 2169.42016601563 2935.36743164063 3473.08666992188 1733.89147949219 3229.69921875 2585.068359375 2645.32885742188 2086.671875 CAR 12:0 6135.099609375 5608.46240234375 4815.53271484375 5131.36083984375 4566.62841796875 6310.77685546875 Guanidinosuccinic Acid 59751.09375 37317.703125 40598.6640625 53842.64453125 67175.9921875 63416.18359375 56617.01953125 61438.27734375 60423.58203125 47224.3515625 40736.4453125 72641.0703125 52909.25 72219.5859375 68911.9609375 63006.83984375 68490.734375 48346.796875 CAR 16:1 75212.125 78178.6640625 69319.6015625 5401.61328125 9294.7197265625 5354.3505859375 5291.11376953125 5754.328125 3886.375 59758.26953125 46452.390625 91834.8984375 4335.12109375 6393.83056640625 5502.0869140625 7065.89453125 5039.06640625 4721.23779296875 CAR 6:0 546229.492431641 465572.90625 459539.237548828 576648.351074219 693938.4375 738313.125 538193.125 671308.377929688 641493.9375 485007.03125 422486.28125 797056.5 593580.0390625 716807.21875 720357.944335938 670710.060058594 689357.75390625 611177.125 Histidine 33144.65625 26716.236328125 22447.716796875 34313.5078125 34424.59765625 39483.703125 30774.3984375 34967.96484375 37853.34765625 22027.615234375 19194.12890625 34641.30078125 33895.88671875 39477.49609375 42500.04296875 43373.76171875 38734.80078125 38015.06640625 Leucine 318703.34375 270316.375 252226.21875 337201.4375 405435 399790.5 292293.53125 363606.3125 371337.6875 274836.25 259414.5625 427964.6875 344044.46875 400192.25 423035.25 414253.25 407105.03125 364201.28125 CAR 18:3 15796.2861328125 16023.5576171875 17854.423828125 12518.5068359375 11032.919921875 18176.048828125 CAR 18:2 219529.53125 203101.078125 193570.875 13762.712890625 20635.294921875 16936.466796875 12242.0419921875 14328.4765625 11174.001953125 186758.90625 131515.78125 261176.796875 10933.724609375 15589.0771484375 13938.9560546875 11942.189453125 12819.0341796875 9611.5439453125 LPC 16:0 3083627.34179688 3247145.5703125 2963629.09375 244618.5625 335227.541503906 246195 205801.828125 211495.609375 176294.296875 2737557.64453125 1999834.97851563 3846078.88671875 182852.25 216675.46875 224199.8125 165040.640625 198295.71875 159476.8125 LPC 16:1 25464.595703125 32802.06640625 29610.662109375 4396.0537109375 6220.18896484375 4461.3662109375 3718.13110351563 3800.42016601563 3565.45458984375 24230.73828125 19672.490234375 25165.736328125 3103.09130859375 3450.96704101563 3040.16528320313 2347.71215820313 2701.53295898438 3034.72338867188 LPC 18:0 3512579.64587402 3527502.06945801 3462138.28149414 471685.59375 554094.75 498426.4375 346809.875 400926.5 333995.78125 3050326.25 2409253.75 3890601.89831543 320414.375 373146.65625 419076.59375 271878.9375 314511.46875 298034.5 LPC 18:1 1528465.03369141 1331411.375 1373192 137097.265625 179178.125 142849.34375 115818.7734375 115069.1640625 107347.3125 1382067.5 1026095.9375 1648431.23486328 110610.2734375 141516.453125 133402 102143.84375 128796.125 97100.4609375 LPC 18:2 894084.875 1330082.375 1198706.125 105380.265625 160420.9375 110219.2578125 76732.734375 102365.3671875 84470.9453125 779546.1875 578017.5 760943.125 42631.8828125 59940.03125 58064.140625 37621.9921875 41386.62890625 46432.453125 LPC 18:3 20993.8671875 20140.056640625 19193.888671875 19687.888671875 13479.4365234375 23418.576171875 LPC 20:0 11413.4775390625 11783.6826171875 10799.7998046875 10149.9013671875 7690.28564453125 10933.0625 LPC 20:1 25024.662109375 25416.197265625 21100.25 3662.23486328125 3648.24731445313 3614.05493164063 3336.66284179688 3420.4560546875 2571.41333007813 20730.794921875 16259.9560546875 25407.365234375 2860.11669921875 3058.42016601563 3082.47509765625 2710.24462890625 2740.228515625 2879.39575195313 CAR 4:1;O2 70715.2265625 56084.87890625 50739.1328125 73325.015625 85633.3984375 99286.859375 67283.8828125 89660.6015625 92245.828125 59001.2578125 49480.41015625 104800.46875 78084.0625 91804.6484375 95972.5 88647.3515625 91563.296875 87730.6484375 N1-Acetylspermidine 9463.3125 6818.201171875 8658.0634765625 7500.970703125 9840.1748046875 10494.65625 5593.02001953125 11038.48828125 8009.599609375 8832.2392578125 8283.0986328125 12138.1806640625 11421.4306640625 9818.935546875 10878.544921875 9003.578125 10048.74609375 10601.47265625 Nicotinamide 9169094 7458620.73193359 7876928.82739258 9069096 10913092 11314559 9531707 10439535 10300028 8395550 7448556 11729794 9289698.13220215 10911588 11068262 10866805 10918942 8710282 CAR 18:1 606948.0625 600953.3125 543481.8125 43121.2109375 57801.33203125 45048.65625 35581.21875 42382.890625 35185.3828125 512999.21875 388465.0625 621292.6875 33996.72265625 39933.95703125 38692.83984375 37098.84765625 39309.30078125 34356.9921875 CAR 16:0 197544 220173.28125 189992.28125 14274.8544921875 22840.48046875 18452.59765625 12582.521484375 16158.90625 11471.6875 166928.734375 124549.6953125 271508.34375 12016.2705078125 14898.7060546875 14845.6220703125 13537.810546875 16534.724609375 9602.208984375 PC 34:2 4241756 5060542.5 4688489.5 1103698.375 1052347.375 1051837.75 791073.625 876779.0625 773523.75 4303156.5 3484290.75 5708756.5 669692.125 782645.8125 954619.75 621646.75 756455 576926.0625 PC 36:2 987510.75 1148874.25 1091579 261730.34375 242849.65625 227991.4375 177283.015625 208408.734375 165664.390625 993566.375 785708 1258629.375 159369.265625 195996.75 218960.953125 148059.6875 170610.28125 132583.046875 PC 36:4 2373798.5 2921615.25 2881676 618403.25 640016.9375 638642.3125 487546.78125 540650.125 468279.0625 2448566 2010136 3058893.75 387906.84375 493745 583567.5625 394177.625 431452.84375 334807.125 PC O-36:4 12961.26171875 16343.8662109375 16955.779296875 3266.69970703125 3335.71630859375 3818.91796875 2757.07568359375 3584.51635742188 2642.7099609375 13656.2822265625 11733.0947265625 18412.94140625 2231.798828125 2920.47729492188 3963.22094726563 1966.04724121094 2123.22485351563 1699.27453613281 PE 40:5 18403.677734375 20887.53515625 20258.9921875 5260.6865234375 4428.9482421875 6007.767578125 4493 5302.6826171875 4136.55029296875 14477.388671875 14624.67578125 21679.697265625 4497.798828125 5182.9453125 6813.41845703125 3876.00073242188 4394.46044921875 3253.85302734375 Pipecolinic Acid 18755.91796875 15483.1044921875 14434.328125 25840.84375 25952.04296875 26742.4921875 20859.376953125 27156.212890625 26470.62890625 18627.76953125 17098.595703125 20896.095703125 23564.68359375 31946.58984375 30362.12109375 29439.703125 28247.107421875 27829.03125 Proline Betaine 1141839.875 934544.875 823834.6875 1129122 1298395.5 1527760.875 998187.0625 1320695.75 1407513.625 987752.375 876048.4375 1552815.375 1242676.375 1514781 1540609.875 1370922.25 1452435.25 1286395.5 CAR 3:0 507170.0625 408979.625 371471.8125 551181.8125 599844.5625 735477.5625 450105.71875 605883.4375 625017.3125 443668.4375 392156.6875 746449.9375 562334 712685.75 734165.3125 626914.8125 621206.5625 602846.3125 S-Adenosylmethionine 1927.03479003906 1414.75646972656 1557.90661621094 2137.94262695313 2124.73095703125 2393.98266601563 1311.14038085938 1972.43286132813 2119.11157226563 1466.8095703125 1396.58435058594 2529.37939453125 1636.99133300781 2049.79296875 2519.88842773438 2173.478515625 2288.21459960938 2100.57421875 SM d34:0 51448.5199082641 47083.3334590149 206680.674762756 14427.0695333863 972.859658355727 11151.4303842163 994.506868438735 81180.5108345643 44738.5704241943 219546.154304687 4018.74247994995 1008.28083480836 987.109763030993 SM d34:1 101952.1328125 128530.421875 125756.53125 17546.677734375 18288.158203125 14695.947265625 11138.416015625 14439.5361328125 10831.931640625 114932.6171875 74801.0703125 155027.09375 11370.23828125 12382.078125 15592.1494140625 8537.072265625 10659.822265625 7275.400390625 CAR 18:0 143111.1875 146072.890625 125417.046875 15190.7744140625 17064.453125 13323.7509765625 11988.6083984375 14791.0322265625 11880.3203125 118371.0859375 89542.5625 135915.046875 9819.080078125 11969.3388671875 12903.4990234375 17426.46484375 10786.87890625 11530.5537109375 CAR 14:1 8723.4052734375 7823.70361328125 7897.93505859375 6704.7861328125 5004.89111328125 9254.587890625 2477.12426757813 CAR 5:0 5678.25374664306 6287.05044607464 CAR 16:0;O 14058.43359375 10540.3203125 10294.8232421875 1818.8896484375 2270.34497070313 2119.28930664063 8684.7734375 7803.74267578125 8222.5595703125 2321.0048828125 2215.61767578125 2279.09936523438 2162.0400390625 CAR 16:1;O 9596.7392578125 10082.7265625 9153.44921875 2094.6171875 8296.6240234375 6568.29833984375 6352.5458984375 SM 36:1;O2 18187.125 22494.353515625 20896.787109375 2741.22900390625 3492.81518554688 2785.8017578125 2115.82788085938 2277.23266601563 2199.46948242188 18710.361328125 11644.6396484375 33014.64453125 2212.05346679688 1915.35461425781 2721.17407226563 1653.89038085938 2097.15844726563 SM 36:2;O2 2991.72021484375 4048.94873046875 3360.50634765625 3246.46606445313 2534.23779296875 5410.98046875 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Formula Measured mass (mean of reversed-phase experiment set 1) Mass accuracy (ppm, mean of reversed-phase experiment set 1) Retention time (mean of reversed-phase experiment set 1) Pubchem ID InChiKeys 2-Aminoadipic Acid C6H11NO4 161.0681 4.35 0.61 469 OYIFNHCXNCRBQI-UHFFFAOYSA-N 3-Methylhistidine C7H11N3O2 169.0848 1.77 0.88 64969 JDHILDINMRGULE-LURJTMIESA-N 5'-Methylthioadenosine C11H15N5O3S 297.0898 0.67 0.88 CAR 2:0 C9H17NO4 203.1166 3.94 0.6 Arginine C6H14N4O2 174.1105 6.89 0.85 6322 ODKSFYDXXFIFQN-BYPYZUCNSA-N Betaine C5H11NO2 117.0794 3.42 0.67 247 KWIUHFFTVRNATP-UHFFFAOYSA-N Bilirubin C33H36N4O6 584.2652 2.91 2.38 5280352 BPYKTIZUTYGOLE-IFADSCNNSA-N CAR 4:0 C11H21NO4 231.1475 1.73 0.61 Choline C5H13NO 103.0998 0.97 0.6 305 OEYIOHPDSNJKLS-UHFFFAOYSA-N Dimethylarginine C8H18N4O2 202.1421 4.45 0.67 123831 YDGMGEXADBMOMJ-LURJTMIESA-N CAR 12:0 C19H37NO4 343.2728 1.46 1.3 Guanidinosuccinic Acid C5H9N3O4 175.0581 6.85 0.6 97856 VVHOUVWJCQOYGG-UHFFFAOYSA-N CAR 16:1 C23H43NO4 397.3171 5.29 2.27 CAR 6:0 C13H25NO4 259.1784 0 0.68 Histidine C6H9N3O2 155.0691 2.58 0.9 6274 HNDVDQJCIGZPNO-YFKPBYRVSA-N Leucine C6H13NO2 131.0936 7.63 0.62 6106 ROHFNLRQFUQHCH-YFKPBYRVSA-N CAR 18:3 C25H43NO4 421.3219 6.41 2.09 CAR 18:2 C25H45NO4 423.335 0.24 2.51 LPC 16:0 C24H50NO7P 495.3329 0.81 2.9 LPC 16:1 C24H48NO7P 493.3177 1.82 2.33 LPC 18:0 C26H54NO7P 523.3639 0.19 3.62 LPC 18:1 C26H52NO7P 521.3484 0.58 3.07 LPC 18:2 C26H50NO7P 519.3326 0.19 2.57 LPC 18:3 C26H48NO7P 517.3171 0.58 2.17 LPC 20:0 C28H58NO7P 551.395 0.18 4.2 LPC 20:1 C28H56NO7P 549.3793 0.18 3.74 CAR 4:1;O2 C11H19NO6 261.1209 1.15 0.65 N1-Acetylspermidine C9H21N3O 187.1683 1.07 0.79 496 MQTAVJHICJWXBR-UHFFFAOYSA-N Nicotinamide C6H6N2O 122.0479 0.82 0.64 936 DFPAKSUCGFBDDF-UHFFFAOYSA-N CAR 18:1 C25H47NO4 425.3503 0.47 3.01 CAR 16:0 C23H45NO4 399.3357 2 2.83 PC 34:2 C42H80NO8P 757.5626 0.53 6.72 PC 36:2 C44H84NO8P 785.5921 1.78 7.73 PC 36:4 C44H80NO8P 781.5631 1.15 6.62 PC O-36:4 C44H82NO7P 767.5781 6.25 7.27 70698779 XWYIIYJIVUGRQE-AMUBJDAWSA-N PE 40:5 C45H80NO8P 793.5693 8.95 6.25 Pipecolinic Acid C6H11NO2 129.0787 2.32 0.61 736316 HXEACLLIILLPRG-RXMQYKEDSA-N Proline Betaine C7H13NO2 143.0948 1.4 0.66 115244 CMUNUTVVOOHQPW-LURJTMIESA-N CAR 3:0 C10H19NO4 217.1313 0.46 0.6 S-Adenosylmethionine C15H22N6O5S 398.1381 2.26 0.88 SM d34:0 C39H81N2O6P 704.5783 6.99 6.36 SM d34:1 C39H79N2O6P 702.5665 1.53 6.11 CAR 18:0 C25H49NO4 427.366 0.47 3.87 CAR 14:1 C21H39NO4 369.2854 6.77 1.57 CAR 5:0 C12H23NO4 245.1611 6.53 0.65 CAR 16:0;O C23H45NO5 415.3288 2.41 2.2 CAR 16:1;O C23H43NO5 413.3114 6.53 1.68 SM 36:1;O2 C41H83N2O6P 730.5982 0.96 6.98 SM 36:2;O2 C41H81N2O6P 728.581 3.02 6.35 METABOLITES_END #END