Summary of Study ST002349

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, where it has been assigned Project ID PR001509. The data can be accessed directly via it's Project DOI: 10.21228/M8N71K This work is supported by NIH grant, U2C- DK119886.


This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002349
Study TitleBiomolecular condensates create phospholipid-enriched microenvironments (Part 1)
Study TypeMetabolomes of in vitro synthesized condensates
Study SummaryProteins 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.
Cornell University
DepartmentDepartment of Pharmacology
LaboratoryDr. Samie Jaffrey
Last NameDumelie
First NameJason
Address1300 York Ave, LC-524, New York City, NY
Submit Date2022-11-04
Raw Data AvailableYes
Raw Data File Type(s)mzdata.xml
Analysis Type DetailOther
Release Date2023-03-01
Release Version2
Jason Dumelie Jason Dumelie application/zip

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Collection ID:CO002584
Collection Summary:Condensate metabolomics. 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 80% methanol to extract metabolites. This method effectively quenches metabolic activity and is well-established for extracting a broad range of metabolites, including polar metabolites77,78. 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. The amount of protein in the pellet was measured using the Quick Start Bradford assay to calculate the metabolites’ protein equivalent mass. Mouse metabolites were initially re-suspended in condensate buffer (50 mM NH4HCO3 pH 7.5, 50 mM NaCl, 1 mM DTT) to a protein equivalent concentration of 938 g/l. The chosen final concentration of metabolites is slightly lower than the 200-300 g/l protein concentration observed in cells79. Metabolites that were not fully soluble in condensate buffer were removed by centrifugation (2x5 min, 16,000 g each), in which only the supernatant was retained. Due to the lack of crowding agents, phase separation required greater concentrations of protein and RNA than typically employed for nucleocapsid and MED1 condensate formation17,32. Purified protein (37.5 μM) was briefly sonicated (10 s) and centrifuged (1 min, 1,000 g) to disrupt any existing condensates and to remove any precipitated proteins. Purified protein (final concentration, 30 μM) was combined with metabolites (final concentration, 150 g/l protein equivalent) and then phage lambda RNA (final concentration, 0.15 μM) in a total volume of 300 µl. An input sample (10 µl) was saved and then the sample was allowed to incubate for 10 min at 25oC. Condensates were then separated from the aqueous environment by centrifugation (10 min, 12,500 g, 25oC). The aqueous phase was removed from the condensate phase and then equal volumes (usually ~ 2 µl) of the aqueous fraction, condensate fraction and input sample were processed for metabolomics using identical approaches as described below. Where shown, representative images of the phases were taken on an iPhone 11. Protein levels in each fraction were evaluated using gels as described above. Where indicated, RNA was added instead to the nucleocapsid at a concentration of either 0 μM or 0.6 μM. In these experiments, all other conditions, including buffer concentrations, were identical to other condensate metabolomics experiments. Notably, in a different subset of experiments, metabolites were added to MED1 condensates after the 10 min incubation rather than prior to the incubation. Metabolite enrichment in these condensates was highly correlated to the other MED1 condensates (r = 0.9, Pearson’s correlation), suggesting that the timing of metabolite addition may not be important. Metabolites were then extracted from each fraction and the input for LC-MS as follows. First the samples were diluted in ammonium bicarbonate buffer (50 mM NH4HCO3 pH 7.5) and briefly heated (2 min, 65oC) to disrupt condensates before being added immediately to 4x volume of ice-cold 100% methanol to precipitate protein and RNA. This heating step does not appear to be necessary for extracting these metabolites and can be excluded (Extended Data Fig. 2e,g). Protein and RNA were separated from metabolites by vortexing the samples (2 min), followed by incubation at -25oC (10 min) and then centrifugation (5 min, 13,000 rpm). The supernatant was saved and the process was repeated on the pellet two more times after adding 200 µl of 80% methanol each time to the pellet. The three supernatants were combined and centrifuged (10 min, 14000 rpm) to remove any additional macromolecules. The final supernatant was collected and dried using a SpeedVac Concentrator run at 25oC.
Sample Type:Liver
Collection Method:80% methanol
Storage Conditions:-80℃