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High-throughput and proteome-wide discovery of endogenous biomolecular condensates

Nature Chemistry (2024)Cite this article

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Abstract

Phase separation inside mammalian cells regulates the formation of the biomolecular condensates that are related to gene expression, signalling, development and disease. However, a large population of endogenous condensates and their candidate phase-separating proteins have yet to be discovered in a quantitative and high-throughput manner. Here we demonstrate that endogenously expressed biomolecular condensates can be identified across a cell’s proteome by sorting proteins across varying oligomeric states. We employ volumetric compression to modulate the concentrations of intracellular proteins and the degree of crowdedness, which are physical regulators of cellular biomolecular condensates. The changes in degree of the partition of proteins into condensates or phase separation led to varying oligomeric states of the proteins, which can be detected by coupling density gradient ultracentrifugation and quantitative mass spectrometry. In total, we identified 1,518 endogenous condensate proteins, of which 538 have not been reported before. Furthermore, we demonstrate that our strategy can identify condensate proteins that respond to specific biological processes.

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Fig. 1: Schematic diagram of high-throughput identification of biomolecular condensates.
Fig. 2: High-throughput identification of phase-separating proteins.
Fig. 3: Validation of condensate proteins identified from volume compression.
Fig. 4: Identification of condensate proteins in response to short-term treatment with TGF-β.
Fig. 5: Identification of condensate proteins in response to long-term treatment with TGF-β.
Fig. 6: Proteome-wide analysis of endogenous-expression phase-separating proteins.

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Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE114 partner repository with the dataset identifier PXD048218. Due to the size and lack of available condensate imaging databases, raw imaging data are available upon request to the corresponding author. All primary data are included in the source data associated with each figure accompanying this paper. Source data are provided with this paper.

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Acknowledgements

We acknowledge financial support from the National Natural Science Foundation of China (grants 32171248 and 12102142 to Y.L., 22074047 and 21775049 to B.-F.L. and 31700746 to P.C.) and the Fundamental Research Funds for Central Universities (HUST no. 2021GCRC056 to Y.L.).

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  1. These authors contributed equally: Pengjie Li, Peng Chen.

Authors and Affiliations

  1. The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics – Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei Province, China

    Pengjie Li, Peng Chen, Fukang Qi, Jinyun Shi, Wenjie Zhu, Jiashuo Li, Peng Zhang, Han Xie, Lina Li, Mengcheng Lei, Xueqing Ren, Wenhui Wang, Liang Zhang, Xufu Xiang, Yiwei Zhang, Zhaolong Gao, Xiaojun Feng, Wei Du, Xin Liu, Bi-Feng Liu & Yiwei Li

  2. Department of Gastroenterology, Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Institute of Liver and Gastrointestinal Diseases, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China

    Limin Xia

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Contributions

Conceptualization was provided by Y.L. and B.-F.L., methodology by Y.L. and P.L., investigations by P.L., P.C., Y.L., H.X., L.L., M.L., X.R., W.W., W.Z., L.Z., X.X., Y.Z. and L.X., visualization by Y.L., P.L., P.C., F.Q., J.S., J.L., P.Z., Z.G., X.F., W.D. and X.L., funding acquisition by Y.L. and B.-F.L., project administration by Y.L. and supervision by Y.L. and B.-F.L. The original draft was written by Y.L. and P.L., and writing, review and editing by Y.L., P.L. and B.-F.L.

Corresponding authors

Correspondence to Bi-Feng Liu or Yiwei Li.

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The authors declare no competing interests.

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Nature Chemistry thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 In situ imaging confirmed that the isolated condensates remained undissociated upon the treatment of the RIPA or NP-40 lysis buffer.

a, Cells were transfected YBX1-EGFP to form YBX1-EGFP condensates in microfluidic channel. The RIPA buffer or NP-40 buffer was introduced into the cells by microchannels. b, YBX1-EGFP condensates remained for at least 40 mins with the treatment of NP-40 buffer. However, NP-40 was observed to insufficiently dissolve cell membrane and be unable to disrupt the nuclear membrane. c, YBX1-EGFP condensates remained for at least 40 mins with the treatment of RIPA buffer, while cellular lipid membrane and nuclear structure were sufficiently dissolved by RIPA buffer. Representative results from three independent experiments.

Extended Data Fig. 2 In situ imaging confirmed that the condensates still retained upon the step of pre-clear centrifugation.

a, Cell lysate was centrifuged by 12000 g, and supernatant was introduced into the cells by microchannels. b, YBX1-EGFP condensates remained after pre-clear centrifugation step. c, YAP1-EGFP condensates remained after pre-clear centrifugation step. Representative results from two independent experiments.

Extended Data Fig. 3 Testing droplet-like behaviors of MRPL23 condensates and TOE1 condensates.

a, Time-dependent images showed the liquid-like fusion and splitting behaviors of the MRPL23-GFP condensates. b, Fluorescent image of MRPL23-GFP condensates. The dash line circle indicated the area of nucleus. c, The percentage of MRPL23-GFP in the condensed form as compared to the total cellular protein. n = 7 independent experiments. d-e, Quantification (d) and sequential images (e) of FRAP assays on MRPL23-GFP condensates showed the recovery of MRPL23-GFP after photo-bleaching. n = 6 independent experiments. f, Time-dependent images showed the liquid-like fusion and splitting behaviors of the TOE1-GFP condensates. g, Fluorescent image of TOE1-GFP condensates. The dash line circle indicated the area of nucleus. h, The percentage of TOE1-GFP in the condensed form as compared to the total cellular protein. n = 10 independent experiments. i-j, Quantification (i) and sequential images (j) of FRAP assays on TOE1-GFP condensates showed the recovery of TOE1-GFP after photo-bleaching. n = 6 independent experiments. In c, d, h, i, data are means ± SD and were analysed by two-sided unpaired student’s t test.

Source data

Extended Data Fig. 4 Testing droplet-like behaviors of ASNS condensates and LTA4H condensates.

a, Time-dependent images showed the liquid-like fusion and splitting behaviors of the ASNS-GFP condensates. b, Fluorescent image of ASNS-GFP condensates. The dash line circle indicated the area of nucleus. c, The percentage of ASNS-GFP in the condensed form as compared to the total cellular protein. n = 7 independent experiments, OE represents overexpression. d-e, Quantification (d) and sequential images (e) of FRAP assays on ASNS-GFP condensates showed the recovery of ASNS-GFP after photo-bleaching. n = 6 independent experiments. f, Time-dependent images showed the liquid-like fusion and splitting behaviors of the LTA4H-GFP condensates. g, Fluorescent image of LTA4H-GFP condensates. The dash line circle indicated the area of nucleus. h, The percentage of LTA4H-GFP in the condensed form as compared to the total cellular protein. n = 7 independent experiments, OE represents overexpression. i-j, Quantification (i) and sequential images (j) of FRAP assays on LTA4H-GFP condensates showed the recovery of LTA4H-GFP after photo-bleaching. n = 6 independent experiments. k, Numbers of detected proteins of 8 types of known membraneless condensates in the detected condensate proteins upon the short-term treatment of TGF-β. In c, d, h, i, data are means ± SD and were analysed by two-sided unpaired student’s t test.

Source data

Extended Data Fig. 5 Testing droplet-like behaviors of DAP3 condensates and IDH1 condensates.

a, Time-dependent images showed the liquid-like fusion and splitting behaviors of the DAP3-GFP condensates. b, Fluorescent image of DAP3-GFP condensates. The dash line circle indicated the area of nucleus. c, The percentage of DAP3-GFP in the condensed form as compared to the total cellular protein. n = 7 independent experiments, OE represents overexpression. d-e, Quantification (d) and sequential images (e) of FRAP assays on DAP3-GFP condensates showed the recovery of ASNS-GFP after photo-bleaching. n = 6 independent experiments. f, Time-dependent images showed the liquid-like fusion and splitting behaviors of the IDH1-GFP condensates. g, Fluorescent image of IDH1-GFP condensates. The dash line circle indicated the area of nucleus. h, The percentage of IDH1-GFP in the condensed form as compared to the total cellular protein. n = 7 independent experiments, OE represents overexpression. i-j, Quantification (i) and sequential images (j) of FRAP assays on IDH1-GFP condensates showed the recovery of IDH1-GFP after photo-bleaching. n = 6 independent experiments. k, Fluorescence images of DAP3-EGFP and IDH1-EGFP transfected into H1975 cells without TGF-β induction (left) and with 2 days of TGF-β treatment (right). Representative results from three independent experiments. l, Numbers of detected proteins of 8 types of known membraneless condensates in the detected condensate proteins upon the long-term treatment of TGF-β. In c, d, h, i, data are means ± SD and were analysed by two-sided unpaired student’s t test.

Source data

Supplementary information

Supplementary Information

Resource table, materials and methods, discussions sections 1–8, Supplementary Figs. 1–30, Tables 1–5, references and raw data of uncropped gel scans.

Reporting Summary

Supplementary Table 1

Results of high-throughput identification of phase separated proteins.

Source data

Source Data Fig. 2

Statistical source data for Figs. 2c, 2d.

Source Data Fig. 3

Statistical source data for Figs. 3e, 3g, 3j, 3k, 3o, 3p.

Source Data Fig. 4

Statistical source data for Figs. 4e, 4g, 4j, 4k, 4o, 4p.

Source Data Fig. 5

Statistical source data for Figs. 5e, 5g, 5j, 5k, 5o, 5p.

Source Data Extended Data Fig. 3

Statistical source data Extended Data Figs. 3c, 3d, 3h, 3i.

Source Data Extended Data Fig. 4

Statistical source data Extended Data Figs. 4c, 4d, 4h, 4i.

Source Data Extended Data Fig. 5

Statistical source data Extended Data Figs. 5c, 5d, 5h, 5i.

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Li, P., Chen, P., Qi, F. et al. High-throughput and proteome-wide discovery of endogenous biomolecular condensates. Nat. Chem. (2024). https://doi.org/10.1038/s41557-024-01485-1

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