#METABOLOMICS WORKBENCH jad2033_20230622_094058 DATATRACK_ID:4109 STUDY_ID:ST002752 ANALYSIS_ID:AN004465 PROJECT_ID:PR001509 VERSION 1 CREATED_ON June 27, 2023, 11:15 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 7 ST:STUDY_TITLE - reversed phase experiment set 2) ST:STUDY_TYPE Metabolomes of in vitro synthesized condensates 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. In this project we used mass spectrometry to ST:STUDY_SUMMARY characterize the metabolomes of condensates. To test this, we prepared mixtures ST:STUDY_SUMMARY of 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. In this ST:STUDY_SUMMARY sub-study, we examined the metabolomes of the mouse liver samples that were used ST:STUDY_SUMMARY to conduct the condensate metabolome experiment described above. 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:PHONE 6465690174 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - RP_experiment_set_2_1 Input_metabolites:mouse liver Used for MS/MS fragmentation=Yes; RAW_FILE_NAME=040323 ZLiverafterRun liver msms neg_P1-E-2_1_409.xml SUBJECT_SAMPLE_FACTORS - RP_experiment_set_2_2 Input_metabolites:mouse liver Used for MS/MS fragmentation=Yes; RAW_FILE_NAME=040323ZLiverafterRUNPOSmsms_P1-E-2_1_407.xml SUBJECT_SAMPLE_FACTORS - RP_experiment_set_2_3 Input_metabolites:mouse liver Used for MS/MS fragmentation=Yes; RAW_FILE_NAME=040623LiverNEG-SPLZ1 PIPE_P1-E-2_1_417.xml SUBJECT_SAMPLE_FACTORS - RP_experiment_set_2_4 Input_metabolites:mouse liver Used for MS/MS fragmentation=Yes; RAW_FILE_NAME=040623LiverNEG-SPLZ1 PS2_P1-E-2_1_425.xml SUBJECT_SAMPLE_FACTORS - RP_experiment_set_2_5 Input_metabolites:mouse liver Used for MS/MS fragmentation=Yes; RAW_FILE_NAME=040623LiverPOS-SPLZ1_P1-E-2_1_413.xml SUBJECT_SAMPLE_FACTORS - RP_experiment_set_2_6 Input_metabolites:Re-run of MED1 Condensate Sample 5 (from study ST002352) Used for MS/MS fragmentation=No; RAW_FILE_NAME=JASON 52b_P1-E-1_1_391.xml SUBJECT_SAMPLE_FACTORS - RP_experiment_set_2_7 Input_metabolites:mouse liver Used for MS/MS fragmentation=No; RAW_FILE_NAME=L1-POS_P1-E-1_1_479.xml SUBJECT_SAMPLE_FACTORS - RP_experiment_set_2_8 Input_metabolites:mouse liver Used for MS/MS fragmentation=No; RAW_FILE_NAME=L2-POS_P1-E-2_1_476.xml SUBJECT_SAMPLE_FACTORS - RP_experiment_set_2_9 Input_metabolites:mouse liver Used for MS/MS fragmentation=No; RAW_FILE_NAME=LiverSPL neg pc sm pi half calibrant_P1-E-2_1_463.xml SUBJECT_SAMPLE_FACTORS - RP_experiment_set_2_10 Input_metabolites:mouse liver Used for MS/MS fragmentation=No; RAW_FILE_NAME=LiverSPL neg pc sm pi_P1-E-1_1_457.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. CO:SAMPLE_TYPE Liver CO:COLLECTION_METHOD 80% methanol CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Metabolites were extracted from mouse livers as discussed in collection. No TR:TREATMENT_SUMMARY special treatment was performed on the mice. #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 were injected (4 µl) SP:SAMPLEPREP_SUMMARY for LC/MS-based targeted metabolite profiling. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatography of metabolites utilized reversed phase chromatography on a CH:CHROMATOGRAPHY_SUMMARY Agilent ZORBAX Eclipse Plus C18, 100 × 2.1 mm, 1.8 μm. Mobile phases consisted CH:CHROMATOGRAPHY_SUMMARY of (A) 10 mM 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 60°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 Model 1290 Infinity II liquid chromatography system CH:COLUMN_NAME Cogent Diamond Hydride (150 × 2.1 mm, 4um) 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 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Impact HD MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE Other MS:ION_MODE POSITIVE MS:MS_COMMENTS The Bruker Impact II QTOF was equipped with a vacuum insulated probe heated MS:MS_COMMENTS electrospray ionization source (VIP-HESI) (Bruker Daltonics, Billerica, USA) to MS:MS_COMMENTS identify representative lipid structures using auto-MS/MS with and without MS:MS_COMMENTS scheduled precursor list fragmentation. Fragments were compared with those MS:MS_COMMENTS deposited in LIPID MAPS, HMDB and MassBank #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Ion abundance (max peak height) MS_METABOLITE_DATA_START Samples RP_experiment_set_2_1 RP_experiment_set_2_2 RP_experiment_set_2_3 RP_experiment_set_2_4 RP_experiment_set_2_5 RP_experiment_set_2_6 RP_experiment_set_2_7 RP_experiment_set_2_8 RP_experiment_set_2_9 RP_experiment_set_2_10 Factors Input_metabolites:mouse liver Input_metabolites:mouse liver Input_metabolites:mouse liver Input_metabolites:mouse liver Input_metabolites:mouse liver Input_metabolites:Re-run of MED1 Condensate Sample 5 (from study ST002352) Input_metabolites:mouse liver Input_metabolites:mouse liver Input_metabolites:mouse liver Input_metabolites:mouse liver PC 34:2 700414 692222 696318 686078 PC O-36:4 90484 10186 7116 59960 SM d34:0 3112 3796 4220 9596 SM d34:1 201538 81174 116556 307152 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Chemical Formula Target Mass Retention time (median) PC 34:2 C42H80NO8P 757.5622 7.6 PC O-36:4 C44H82NO7P 767.5829 8.1 SM d34:0 C39H81N2O6P 704.583225 7.3 SM d34:1 C39H79N2O6P 702.567575 6.9 METABOLITES_END #END