#METABOLOMICS WORKBENCH jad2033_20221116_083951 DATATRACK_ID:3577 STUDY_ID:ST002353 ANALYSIS_ID:AN004099 PROJECT_ID:PR001509 VERSION 1 CREATED_ON November 17, 2022, 11:36 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 3) 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 Synthetic sample #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 Library 100 nM Aqueous Sample 1 metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:aqueous RAW_FILE_NAME=L1 Top.mzdata.xml;p_L1 Top.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 100 nM Condensate Sample 1 metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:condensate RAW_FILE_NAME=L1 Bottom.mzdata.xml;p_L1 Bottom.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 100 nM Input Sample 1 metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:input RAW_FILE_NAME=L1 Input.mzdata.xml;p_L1 Input.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 100 nM Aqueous Sample 2 metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:aqueous RAW_FILE_NAME=L2 Top.mzdata.xml;p_L2 Top.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 100 nM Condensate Sample 2 metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:condensate RAW_FILE_NAME=L2 Bottom.mzdata.xml;p_L2 Bottom.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 100 nM Input Sample 2 metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:input RAW_FILE_NAME=L2 Input.mzdata.xml;p_L2 Input.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 100 nM Aqueous Sample 3 metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:aqueous RAW_FILE_NAME=L3 Top.mzdata.xml;p_L3 Top.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 100 nM Condensate Sample 3 metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:condensate RAW_FILE_NAME=L3 Bottom.mzdata.xml;p_L3 Bottom.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 100 nM Input Sample 3 metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:input RAW_FILE_NAME=L3 Input.mzdata.xml;p_L3 Input.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 1 uM Aqueous Sample 1 metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous RAW_FILE_NAME=M1 Top.mzdata.xml;p_M1 Top.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 1 uM Condensate Sample 1 metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:condensate RAW_FILE_NAME=M1 Bottom.mzdata.xml;p_M1 Bottom.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 1 uM Input Sample 1 metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:input RAW_FILE_NAME=M1 Input.mzdata.xml;p_M1 Input.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 1 uM Aqueous Sample 2 metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous RAW_FILE_NAME=M2 Top.mzdata.xml;p_M2 Top.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 1 uM Condensate Sample 2 metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:condensate RAW_FILE_NAME=M2 Bottom.mzdata.xml;p_M2 Bottom.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 1 uM Input Sample 2 metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:input RAW_FILE_NAME=M2 Input.mzdata.xml;p_M2 Input.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 1 uM Aqueous Sample 3 metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous RAW_FILE_NAME=M3 Top.mzdata.xml;p_M3 Top.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 1 uM Condensate Sample 3 metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:condensate RAW_FILE_NAME=M3 Bottom.mzdata.xml;p_M3 Bottom.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 1 uM Input Sample 3 metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:input RAW_FILE_NAME=M3 Input.mzdata.xml;p_M3 Input.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 10 uM Aqueous Sample 1 metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous RAW_FILE_NAME=H1 Top.mzdata.xml;p_H1 Top.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 10 uM Condensate Sample 1 metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:condensate RAW_FILE_NAME=H1 Bottom.mzdata.xml;p_H1 Bottom.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 10 uM Input Sample 1 metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:input RAW_FILE_NAME=H1 Input.mzdata.xml;p_H1 Input.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 10 uM Aqueous Sample 2 metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous RAW_FILE_NAME=H2 Top.mzdata.xml;p_H2 Top.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 10 uM Condensate Sample 2 metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:condensate RAW_FILE_NAME=H2 Bottom.mzdata.xml;p_H2 Bottom.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 10 uM Input Sample 2 metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:input RAW_FILE_NAME=H2 Input.mzdata.xml;p_H2 Input.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 10 uM Aqueous Sample 3 metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous RAW_FILE_NAME=H4Top.mzdata.xml;p_H4Top.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 10 uM Condensate Sample 3 metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:condensate RAW_FILE_NAME=H4Bottom.mzdata.xml;p_H4Bottom.mzdata.xml SUBJECT_SAMPLE_FACTORS - MED1 Library 10 uM Input Sample 3 metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:input RAW_FILE_NAME=H4Input.mzdata.xml;p_H4Input.mzdata.xml #COLLECTION CO:COLLECTION_SUMMARY Chemical library to perform condensate metabolomics with a defined set of CO:COLLECTION_SUMMARY lipids. To analyze a defined set of lipids at known concentrations, the CO:COLLECTION_SUMMARY following molecules were purchased: phosphatidylcholine (3:0/3:0)(Cayman CO:COLLECTION_SUMMARY Chemical, 32703), phosphatidylcholine (9:0/9:0)(Cayman Chemical, 10009874), CO:COLLECTION_SUMMARY phosphatidylcholine (12:0/12:0)(Echelon Biosciences, L-1112), CO:COLLECTION_SUMMARY phosphatidylcholine (16:0/16:0)(Echelon Biosciences, L-1116), CO:COLLECTION_SUMMARY phosphatidylcholine (18:0/18:0)(Echelon Biosciences, L-1118), CO:COLLECTION_SUMMARY sn-glycero-3-phosphocholine, lysophosphatidylcholine (16:0)(Echelon Biosciences, CO:COLLECTION_SUMMARY L-1516), palmitic acid (Sigma Aldrich, P5585), phosphatidylethanolamine CO:COLLECTION_SUMMARY (16:0/16:0)(Avanti Polar Lipids, 850705), phosphatidylglycerol CO:COLLECTION_SUMMARY (16:0/16:0)(Avanti Polar Lipids, 840455), phosphatidylinositol CO:COLLECTION_SUMMARY (16:0/16:0)(Echelon Biosciences, P-0016), PIP2 (16:0/16:0)(Echelon Biosciences, CO:COLLECTION_SUMMARY P-4516), phosphatidylserine (16:0/16:0)(Echelon Biosciences, L-3116). These CO:COLLECTION_SUMMARY molecules were first dissolved in an appropriate organic solvent and then either CO:COLLECTION_SUMMARY 0.33 pmoles, 3.3 pmoles, or 33 pmoles of each molecule were combined in an CO:COLLECTION_SUMMARY eppendorf tube. The organic solvents were removed using a SpeedVac Concentrator CO:COLLECTION_SUMMARY (Savant, SPD131DDA) at 25oC and the dried chemical libraries were stored at CO:COLLECTION_SUMMARY -80oC. Each tube containing a chemical library was used to perform a single CO:COLLECTION_SUMMARY condensate metabolomics experiment. These libraries were initially re-suspended CO:COLLECTION_SUMMARY in condensate buffer (50 mM NH4HCO3 pH 7.5, 50 mM NaCl, 1 mM DTT). Molecules CO:COLLECTION_SUMMARY that were not fully soluble in condensate buffer were removed by centrifugation CO:COLLECTION_SUMMARY (2x5 min, 16,000 g each), in which only the supernatant was retained. Due to the CO:COLLECTION_SUMMARY lack of crowding agents, phase separation required greater concentrations of CO:COLLECTION_SUMMARY protein and RNA than typically employed for nucleocapsid and MED1 condensate CO:COLLECTION_SUMMARY formation17,32. Purified protein (37.5 μM) was centrifuged (1 min, 1,000 g) to CO:COLLECTION_SUMMARY disrupt any existing condensates and to remove any precipitated proteins. CO:COLLECTION_SUMMARY Purified protein (final concentration, 30 μM) was combined with metabolites CO:COLLECTION_SUMMARY (final concentration, 150 g/l protein equivalent) and then phage lambda RNA CO:COLLECTION_SUMMARY (final concentration, 0.15 μM) in a total volume of 300 µl. An input sample CO:COLLECTION_SUMMARY (10 µl) was saved and then the sample was allowed to incubate for 10 min at CO:COLLECTION_SUMMARY 25oC. Condensates were then separated from the aqueous environment by CO:COLLECTION_SUMMARY centrifugation (10 min, 12,500 g, 25oC). The aqueous phase was removed from the CO:COLLECTION_SUMMARY condensate phase and then equal volumes (usually ~ 2 µl) of the aqueous CO:COLLECTION_SUMMARY fraction, condensate fraction and input sample were processed for metabolomics CO:COLLECTION_SUMMARY using identical approaches as described below. First the samples were diluted in CO:COLLECTION_SUMMARY ammonium bicarbonate buffer (50 mM NH4HCO3 pH 7.5) and briefly heated (2 min, CO:COLLECTION_SUMMARY 65oC) to disrupt condensates before being added immediately to 4x volume of CO:COLLECTION_SUMMARY ice-cold 100% methanol to precipitate protein and RNA. Protein and RNA were CO:COLLECTION_SUMMARY separated from metabolites by vortexing the samples (2 min), followed by CO:COLLECTION_SUMMARY 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. CO:SAMPLE_TYPE Synthetic Mixture CO:COLLECTION_METHOD 80% methanol CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Chemical libraries were added with individual molecules at a concentration of TR:TREATMENT_SUMMARY 100 nM, 1 uM or 10 uM to the condensate-forming low-complexity domain MED1 TR:TREATMENT_SUMMARY tagged with mCherry. Condensates were stimulated with 150 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. #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. Protein and RNA were separated from metabolites by vortexing SP:SAMPLEPREP_SUMMARY the samples (2 min), followed by incubation at -25oC (10 min) and then SP:SAMPLEPREP_SUMMARY centrifugation (5 min, 13,000 rpm). The supernatant was saved and the process SP:SAMPLEPREP_SUMMARY was repeated on the pellet two more times after adding 200 µl of 80% methanol SP:SAMPLEPREP_SUMMARY each time to the pellet. The three supernatants were combined and centrifuged SP:SAMPLEPREP_SUMMARY (10 min, 14000 rpm) to remove any additional macromolecules. The final SP:SAMPLEPREP_SUMMARY supernatant was collected and dried using a SpeedVac Concentrator run at 25oC. SP:SAMPLEPREP_SUMMARY On the day of metabolite analysis, dried-down extracts were reconstituted in 150 SP:SAMPLEPREP_SUMMARY µl 70% acetonitrile, at a relative protein concentration of ~ 2 µg/µl, and 4 SP:SAMPLEPREP_SUMMARY µl of this reconstituted extract was injected for LC/MS-based untargeted SP:SAMPLEPREP_SUMMARY metabolite 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 Library 100 nM Condensate Sample 1 MED1 Library 100 nM Condensate Sample 2 MED1 Library 100 nM Condensate Sample 3 MED1 Library 1 uM Condensate Sample 1 MED1 Library 1 uM Condensate Sample 2 MED1 Library 1 uM Condensate Sample 3 MED1 Library 10 uM Condensate Sample 1 MED1 Library 10 uM Condensate Sample 2 MED1 Library 10 uM Condensate Sample 3 MED1 Library 100 nM Input Sample 1 MED1 Library 100 nM Input Sample 2 MED1 Library 100 nM Input Sample 3 MED1 Library 1 uM Input Sample 1 MED1 Library 1 uM Input Sample 2 MED1 Library 1 uM Input Sample 3 MED1 Library 10 uM Input Sample 1 MED1 Library 10 uM Input Sample 2 MED1 Library 10 uM Input Sample 3 MED1 Library 100 nM Aqueous Sample 1 MED1 Library 100 nM Aqueous Sample 2 MED1 Library 100 nM Aqueous Sample 3 MED1 Library 1 uM Aqueous Sample 1 MED1 Library 1 uM Aqueous Sample 2 MED1 Library 1 uM Aqueous Sample 3 MED1 Library 10 uM Aqueous Sample 1 MED1 Library 10 uM Aqueous Sample 2 MED1 Library 10 uM Aqueous Sample 3 Factors metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:condensate metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:condensate metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:condensate metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:condensate metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:condensate metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:condensate metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:condensate metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:condensate metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:condensate metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:input metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:input metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:input metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:input metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:input metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:input metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:input metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:input metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:input metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:aqueous metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:aqueous metabolite_source:100 nM library | protein:MED1 | RNA:150 nM | fraction:aqueous metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous metabolite_source:1 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous metabolite_source:10 uM library | protein:MED1 | RNA:150 nM | fraction:aqueous sn-glycero-3-phosphocholine 95048.1174072264 100224.872705688 94638.0192089843 1284574.05557764 1138342.6783078 1241890.90176514 16152886.5060031 11211011.2303279 19758735.5782851 96724.2498474121 128106.919326782 79610.6025149535 765380.873909913 966822.029629639 1342419.20349756 12854863.5969029 12104538.220179 18744314.078145 95444.5336502074 201480.050327698 188447.506175415 854459.85141803 998939.11391327 1395907.91194153 12754543.8810164 11861698.8723758 25554795.557002 PC(3:0/3:0) 37904.509345703 36516.058303833 36904.7085583496 435969.308160858 409727.241641968 375971.15836731 4348339.40582068 3243430.39630319 3978967.42426336 44342.8102616882 59421.6902583007 26269.0831956787 380699.29848938 485214.806607056 635313.734433104 6096978.72825327 5983089.07500931 5853145.72592835 37179.5102670289 47297.159460144 41825.835955963 424408.876849976 475930.249510529 685970.636085021 6116260.57893403 5621820.67922723 7371086.27663526 LysoPC(16:0) 130813.178357239 108118.949953613 70720.796734314 41352519.0267138 35414893.84885 28296206.7980412 356944124.603669 435943882.399087 310478501.820921 49503.799115448 16682.1495372009 4685.44190460212 98569.4470388795 144878.740387085 200547.765993195 7450005.86096247 9603030.33540649 5121315.06491541 9720.59903503411 77333.0235061035 106468.583589477 89486.9098010254 53820.6524229431 88581.7087901308 2613152.02766937 2872667.96398102 2571715.10457385 PC(9:0/9:0) 169286.358966613 74103.6051660155 52645.9196926117 9805265.98941798 6724835.57763402 2471604.33208551 79530021.1065523 81412153.8830489 90731874.4491328 7842.5623271179 7418.34326606752 6456.30544938665 69563.4439321136 115108.179079773 160099.97526944 3327009.74171201 3434332.92976468 3067870.11326956 7081.10449246219 5102.60064677428 5464.27256140138 110400.513964142 79514.9236986388 140775.839024353 3654019.18078528 2422723.34535745 7047426.59050036 PC(12:0/12:0) 7442421.29723936 6055102.28189304 1538804.88001962 67104304.4178854 69489902.3530198 62738581.332774 127223011.144884 130602357.596052 133940789.245777 49833.4628390026 46918.6545793552 16313.4282150269 615105.526557139 985794.858529677 901209.199012718 13269152.9566235 16946670.8407844 8986403.09486015 26659.0130587854 23280.9157515239 16535.9216845894 408819.044412792 192067.290703156 270796.495883645 3928333.70631377 5512871.45920262 3212858.50196454 PC(16:0/16:0) 4076404.5842821 3796470.93909265 2586286.55728192 26731786.7667313 29279957.5035482 25159713.3167744 34338052.7869029 31498700.8792181 41900980.813126 19555.0864036236 29225.6973941564 10278.0444830208 293387.366582577 563181.024513028 249026.606534677 5915661.71989481 7155938.18439772 6020717.88453119 135 5850.23677630229 11308.8708893365 32134.2056327476 11279.0395979595 26739.9993406743 2544393.68023555 3948402.39621762 2867495.27110053 PC(18:0/18:0) 130472.454877377 60986.4189372309 97814.0409411772 222811.057786125 311196.965478634 223203.20970137 754937.140845665 498232.220667358 581956.784154064 135 512.376852874763 135 135 9805.08677406595 1235.23716801451 172321.356853958 200409.572277885 178871.444849998 135 135 135 135 135 135 92052.3405347557 161374.774243339 88524.0946618654 PE(16:0/16:0) 573485.63262071 682646.408398283 573945.728227174 476639.618831216 490774.860759527 402820.379565217 163814.138229065 61308.2699658203 17821.8550848388 135 135 135 135 135 135 135 135 135 135 135 135 135 135 135 135 135 135 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Mass RT KEGG Formula PubChem CAS sn-glycero-3-phosphocholine 257.1032 14.81 C00670 C8H21NO6P 3939 28319-77-9 PC(3:0/3:0) 370.1597 11.77 C14H28NO8P 24779217 66414-33-3 LysoPC(16:0) 495.3336 10.18 C24H50NO7P 460602 17364-16-8 PC(9:0/9:0) 510.3154 9.41 C26H52NO8P 3082274 27869-45-0 PC(12:0/12:0) 622.4417 8.81 C32H64NO8P 512874 18194-25-7 PC(16:0/16:0) 733.5636 8.47 D03585 C40H80NO8P 160339 63-89-8 PC(18:0/18:0) 790.6277 8.37 C44H88NO8P 94190 816-94-4 PE(16:0/16:0) 691.507 5.47 C37H74NO8P 445468 923-61-5 METABOLITES_END #END