{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002751","ANALYSIS_ID":"AN004464","VERSION":"1","CREATED_ON":"02-08-2024"},

"PROJECT":{"PROJECT_TITLE":"Biomolecular condensates create phospholipid-enriched microenvironments","PROJECT_TYPE":"Metabolomics of in vitro condensates","PROJECT_SUMMARY":"Proteins and RNA are able to phase separate from the aqueous cellular environment to form sub-cellular compartments called condensates. This process results in a protein-RNA mixture that is chemically distinct from the surrounding aqueous phase. Here we use mass spectrometry to characterize the metabolomes of condensates. To test this, we prepared mixtures of phase-separated proteins and cellular metabolites and identified metabolites enriched in the condensate phase. These proteins included SARS-CoV-2 nucleocapsid, as well as low complexity domains of MED1 and HNRNPA1.","INSTITUTE":"Cornell University","DEPARTMENT":"Department of Pharmacology","LABORATORY":"Dr. Samie Jaffrey","LAST_NAME":"Dumelie","FIRST_NAME":"Jason","ADDRESS":"1300 York Ave, LC-524, New York City, NY","EMAIL":"jdumes98@gmail.com","PHONE":"6465690174","FUNDING_SOURCE":"This work was supported by the National Institutes of Health grants R35NS111631 and R01CA186702 (S.R.J.); R01AR076029, R21ES032347 and R21NS118633 (Q.C.); and NIH P01 HD067244 and support from the Starr Cancer Consortium I13-0037 (S.S.G.).","PUBLICATIONS":"Under revision","DOI":"http://dx.doi.org/10.21228/M8N71K","CONTRIBUTORS":"Jason G. Dumelie, Qiuying Chen, Dawson Miller, Nabeel Attarwala, Steven S. Gross and Samie R. Jaffrey1"},

"STUDY":{"STUDY_TITLE":"Biomolecular condensates create phospholipid-enriched microenvironments (Part 5)","STUDY_TYPE":"Metabolomes of mouse liver","STUDY_SUMMARY":"In this study we used LC-MS and MS/MS to characterize the metabolomes of the input mouse liver metabolites used in the first two studies of this submission.","INSTITUTE":"Cornell University","DEPARTMENT":"Department of Pharmacology","LABORATORY":"Dr. Samie Jaffrey","LAST_NAME":"Dumelie","FIRST_NAME":"Jason","ADDRESS":"1300 York Ave, LC-524, New York City, NY","EMAIL":"srj2003@med.cornell.edu","PHONE":"6465690174","SUBMIT_DATE":"2023-06-14"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"ANP_experiment_set_3_replicate_2",
"Factors":{"Factor":"2 μl"},
"Additional sample data":{"RAW_FILE_NAME":"jason liver anpneg1.mzdata.xml jason liver anppos1.mzdata.xml"}
},
{
"Subject ID":"-",
"Sample ID":"ANP_experiment_set_3_replicate_3",
"Factors":{"Factor":"2 μl"},
"Additional sample data":{"RAW_FILE_NAME":"jason liver anpneg1 2.mzdata.xml jason liver anppos1 2.mzdata.xml"}
},
{
"Subject ID":"-",
"Sample ID":"ANP_experiment_set_3_replicate_4",
"Factors":{"Factor":"2 μl"},
"Additional sample data":{"RAW_FILE_NAME":"jason liver anpneg1 3.mzdata.xml jason liver anppos1 3.mzdata.xml"}
},
{
"Subject ID":"-",
"Sample ID":"ANP_experiment_set_3_replicate_5",
"Factors":{"Factor":"2 μl"},
"Additional sample data":{"RAW_FILE_NAME":"jason liver anpneg1 4.mzdata.xml jason liver anppos1 4.mzdata.xml"}
},
{
"Subject ID":"-",
"Sample ID":"ANP_experiment_set_3_replicate_6",
"Factors":{"Factor":"2 μl"},
"Additional sample data":{"RAW_FILE_NAME":"jason liver anpneg2.mzdata.xml jason liver anppos2.mzdata.xml"}
},
{
"Subject ID":"-",
"Sample ID":"ANP_experiment_set_3_replicate_7",
"Factors":{"Factor":"2 μl"},
"Additional sample data":{"RAW_FILE_NAME":"Liver NEG1.mzdata.xml"}
},
{
"Subject ID":"-",
"Sample ID":"ANP_experiment_set_3_replicate_8",
"Factors":{"Factor":"2 μl"},
"Additional sample data":{"RAW_FILE_NAME":"Liver NEG2.mzdata.xml"}
},
{
"Subject ID":"-",
"Sample ID":"ANP_experiment_set_3_replicate_1",
"Factors":{"Factor":"4 μl"},
"Additional sample data":{"RAW_FILE_NAME":"liver1 ANPPOS MS.mzdata.xml"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Mouse metabolites were collected from the liver of female mice using methanol extraction. After euthanizing a mouse, the liver was immediately frozen in liquid nitrogen. We then used cold (-20C or colder) 80% methanol to extract metabolites. First, 1 ml of 80% methanol was added to the liver and incubated for 10 min at -20oC. Glass beads were added to the liver and then the liver was lysed by bead-beating for 45 s using a Tissuelyser cell disrupter (Qiagen). The lysate was incubated for 10 min at -20oC and centrifuged (13200 rpm, 5 min) to separate metabolites from macromolecules. The supernatant was collected and 200 µl of 80% methanol was added to the pellet. The incubation, shaking and centrifugation steps were repeated twice to extract more metabolites from the pellet. The three supernatants were combined and centrifuged (14000 rpm, 10 min) to separate any remaining macromolecules from the metabolites. The combined supernatants were dried using a SpeedVac Concentrator (Savant, SPD131DDA) at 25oC and the dried metabolite samples were stored at -80oC.","SAMPLE_TYPE":"Liver","COLLECTION_METHOD":"80% methanol","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"All samples were treated identically for this sub-study, except a higher volume (4 μl ANP_experiment_set_3_replicate_1) of one sample was injected than for the other samples (2 μl)."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"On the day of metabolite analysis, dried-down extracts were reconstituted in 150 µl 70% acetonitrile, at a relative protein concentration of ~ 2 µg/µl. Either 4 µl (replicate 1) or 2 µl (all other replicates) of this reconstituted extract was injected for LC/MS-based targeted and untargeted metabolite profiling.","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Tissue extracts were analyzed by LC/MS as described previously, using a platform comprised of an Agilent Model 1290 Infinity II liquid chromatography system coupled to an Agilent 6550 iFunnel time-of-flight MS analyzer. Chromatography of metabolites utilized aqueous normal phase (ANP) chromatography on a Diamond Hydride column (Microsolv). Mobile phases consisted of: (A) 50% isopropanol, containing 0.025% acetic acid, and (B) 90% acetonitrile containing 5 mM ammonium acetate. To eliminate the interference of metal ions on chromatographic peak integrity and electrospray ionization, EDTA was added to the mobile phase at a final concentration of 5 µM. The following gradient was applied: 0-1.0 min, 99% B; 1.0-15.0 min, to 20% B; 15.0 to 29.0, 0% B; 29.1 to 37min, 99% B.","INSTRUMENT_NAME":"Agilent Model 1290 Infinity II liquid chromatography system","COLUMN_NAME":"Cogent Diamond Hydride (150 × 2.1 mm, 4um)","COLUMN_TEMPERATURE":"26","FLOW_GRADIENT":"The following gradient was applied: 0-1.0 min, 99% B; 1.0-15.0 min, to 20% B; 15.0 to 29.0, 0% B; 29.1 to 37min, 99% B.","FLOW_RATE":"0.3 mL/min","SOLVENT_A":"50% isopropanol/50% water; 0.025% acetic acid","SOLVENT_B":"90% acetonitrile/10% water; 5 mM ammonium acetate","CHROMATOGRAPHY_TYPE":"Normal phase"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Agilent 6550 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"Other","MS_COMMENTS":"LC/MS-based targeted and untargeted metabolite profiling. For targeted analysis, raw LC/MS data was extracted by MassProfinder 8.0 (Agilent Technologies) using an in-house annotated personal metabolite database that contains 863 metabolites (Agilent Technologies). Additionally, molecular feature extraction (MFE) was performed for untargeted metabolite profiling using MassProfinder 8.0 (Agilent Technologies). The untargeted molecular features were imported into MassProfiler Professional 15.1 (MPP, Agilent Technologies) and searched against Metlin personal metabolite database (PCDL database 8.0), Human Metabolome Database (HMDB) and an in-house phospholipid database for tentative metabolite ID assignments, based on monoisotopic neutral mass (< 5 ppm mass accuracy) matches. Furthermore, a molecular formula generator (MFG) algorithm in MPP was used to generate and score empirical molecular formulae, based on a weighted consideration of monoisotopic mass accuracy, isotope abundance ratios, and spacing between isotope peaks. A tentative compound ID was assigned when PCDL database and MFG scores concurred for a given candidate molecule. Tentatively assigned molecules were reextracted using Profinder 8.0 for confirmation of untargeted results. Only non-lipid metabolites that were identified in study ST002349 were retained for this analysis.","ION_MODE":"NEGATIVE"},

"MS_METABOLITE_DATA":{
"Units":"Ion abundance (peak area)",

"Data":[{"Metabolite":"1-Methyladenosine","ANP_experiment_set_3_replicate_2":"109709.5102","ANP_experiment_set_3_replicate_3":"","ANP_experiment_set_3_replicate_4":"26107.4893","ANP_experiment_set_3_replicate_5":"26993.0664","ANP_experiment_set_3_replicate_6":"126498.8311","ANP_experiment_set_3_replicate_7":"","ANP_experiment_set_3_replicate_8":"","ANP_experiment_set_3_replicate_1":""},{"Metabolite":"2-Aminoadipic Acid","ANP_experiment_set_3_replicate_2":"338182.2768","ANP_experiment_set_3_replicate_3":"594052.9272","ANP_experiment_set_3_replicate_4":"694711.4965","ANP_experiment_set_3_replicate_5":"706744.8100","ANP_experiment_set_3_replicate_6":"373184.4424","ANP_experiment_set_3_replicate_7":"","ANP_experiment_set_3_replicate_8":"","ANP_experiment_set_3_replicate_1":""},{"Metabolite":"2-Aminoisobutyric Acid","ANP_experiment_set_3_replicate_2":"472753.9855","ANP_experiment_set_3_replicate_3":"750479.4606","ANP_experiment_set_3_replicate_4":"587674.5457","ANP_experiment_set_3_replicate_5":"","ANP_experiment_set_3_replicate_6":"404002.1815","ANP_experiment_set_3_replicate_7":"43785.7974","ANP_experiment_set_3_replicate_8":"46190.5825","ANP_experiment_set_3_replicate_1":""},{"Metabolite":"2-Hydroxybutyric Acid","ANP_experiment_set_3_replicate_2":"7809206.5917","ANP_experiment_set_3_replicate_3":"11193064.3419","ANP_experiment_set_3_replicate_4":"7449349.1614","ANP_experiment_set_3_replicate_5":"","ANP_experiment_set_3_replicate_6":"5108083.3952","ANP_experiment_set_3_replicate_7":"1786414.3668","ANP_experiment_set_3_replicate_8":"1727489.6895","ANP_experiment_set_3_replicate_1":""},{"Metabolite":"2-Hydroxyphenylacetic Acid","ANP_experiment_set_3_replicate_2":"","ANP_experiment_set_3_replicate_3":"","ANP_experiment_set_3_replicate_4":"","ANP_experiment_set_3_replicate_5":"","ANP_experiment_set_3_replicate_6":"","ANP_experiment_set_3_replicate_7":"","ANP_experiment_set_3_replicate_8":"","ANP_experiment_set_3_replicate_1":""},{"Metabolite":"2-Methylcitric Acid","ANP_experiment_set_3_replicate_2":"896949.4999","ANP_experiment_set_3_replicate_3":"1379885.8030","ANP_experiment_set_3_replicate_4":"1256760.4565","ANP_experiment_set_3_replicate_5":"","ANP_experiment_set_3_replicate_6":"2010424.2274","ANP_experiment_set_3_replicate_7":"217020.9832","ANP_experiment_set_3_replicate_8":"190410.4398","ANP_experiment_set_3_replicate_1":""},{"Metabolite":"3-Aminoisobutyric 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