#METABOLOMICS WORKBENCH jad2033_20221116_083951 DATATRACK_ID:3577 STUDY_ID:ST002353 ANALYSIS_ID:AN004100 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 NEGATIVE 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 Palmitic acid 6693564.92929273 5494993.84635132 5599044.81421887 7031405.05302905 7983906.70946338 7429736.17232714 4248661.82858826 23501745.2064829 4655878.36239257 6585645.56416187 7424679.71875976 3899044.16138721 5147074.2941438 6537753.67539062 5668873.72906615 5654823.28255933 5407585.03264941 4736340.54955371 1721990.00828482 8208369.77895751 4908535.44942774 4886665.40209497 7072455.95190234 5266215.58012744 6032025.55395434 4901324.15898828 3614481.53658008 PG(16:0/16:0) 2435901.03053126 15862523.8861784 1921368.98833072 7269052.24347144 10713552.6895577 8467777.38671537 12690592.2110736 28268839.2302003 3567491.48828467 11550.9634884643 104616.588147644 1616.67685302734 102543.157090576 333718.043959621 109966.07760357 2951927.01128131 2798376.63380928 523408.615584473 3575.99591552735 6500.07313705445 2552.85514343262 6827.66313659666 8910.49635253907 9159.00347473144 872771.769761949 1098148.95098544 185796.1005961 PI(16:0/16:0) 600080.237881041 474169.917394897 650607.584706543 810807.04700354 1850615.228224 1403043.69323682 3051339.36485167 4336059.86954197 1057917.1677373 135 2729.66770996094 135 135 34095.5964056395 13843.2575778198 742306.991349365 592210.669806639 315628.770388062 135 1999.77688568115 135 135 135 135 42131.8783614502 35084.4988143921 3380.92217285156 PI(45)P2(16:0/16:0) 26419.2188843031 31711.2590063486 7455.39615516669 135245.421676875 144442.933435959 140572.018490839 533350.95619464 594528.428129264 475363.168191987 3480.77297943114 1249.07403182982 1888.40922637939 2935.0774702453 5557.94729766837 6082.01652626034 34649.2142657471 55767.6047261152 27920.2948535919 2069.8720770264 929.394803695667 1149.76533233644 3173.29736991884 542.409973144539 2922.63057281493 21128.9935900818 19464.0623405457 23130.3872657776 PS(16:0/16:0) 1171478.55195834 1003055.57668415 1387078.35885182 963043.248274567 1139715.78876666 921553.795465729 1675004.95612523 1573612.54815022 179971.826880859 2792.46126586914 79650.9921885681 135 135 18605.3902191162 13830.433343811 73393.8294247742 44762.4239234923 28788.6928268433 3941.0306490326 3634.21515136719 1990.24612335205 135 5354.86334579468 10938.7723571777 13892.0876812744 17286.730330658 4891.36015151978 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Mass RT KEGG Formula PubChem CAS Palmitic acid 256.2411 1.45 C00249 C16H32O2 985 57-10-3 PG(16:0/16:0) 722.5083 2.5 C38H75O10P 11846227 200880-41-7 PI(16:0/16:0) 810.5292 2.73 C41H79O13P 24779556 34290-57-8 PI(45)P2(16:0/16:0) 970.4482 9.04 C41H78O19P3 1628353-01-4 PS(16:0/16:0) 735.5096 2.56 C38H73NO10P 46891791 145849-32-7 METABOLITES_END #END