ReviewNature protocols2026
High-throughput identification of endogenous biomolecular condensates and phase-separating proteins.
Pengjie Li, Fukang Qi, Wenjie Zhu, Jiashuo Li, Jinyun Shi, Xinyu Tu, Mengran Wang, Peng Chen, Bi-Feng Liu, Yiwei Li
Abstract readReview
In one paragraphReview in Nature protocols, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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5 · Who and what moneyAuthors and funding
10 authors.
Pengjie LiKey Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.ORCID http://orcid.org/0000-0002-8057-368X Fukang QiKey Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.ORCID http://orcid.org/0009-0008-6860-9847 Wenjie ZhuKey Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
Jiashuo LiKey Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
Jinyun ShiKey Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
Xinyu TuKey Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
Mengran WangKey Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
Peng ChenKey Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
Bi-Feng LiuKey Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China. bfliu@mail.hust.edu.cn.ORCID http://orcid.org/0000-0002-2135-0873 Yiwei LiKey Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China. yiweili@hust.edu.cn.ORCID http://orcid.org/0000-0002-5203-0290 Funding
National Natural Science Foundation of China (National Science Foundation of China) 13102142National Natural Science Foundation of China (National Science Foundation of China) 32171248
6 · The paper itselfAbstract
Biomolecular condensates formed through liquid-liquid phase separation regulate cellular processes, and their dysregulation causes disease. Current methods for identifying endogenous phase-separating proteins have low throughput and cannot capture dynamic responses to stimuli. Here we present a protocol combining osmotic compression or transforming growth factor-β (TGF-β) treatment to induce condensation with sucrose density gradient centrifugation and quantitative mass spectrometry to enable systematic, high-throughput identification of endogenous condensates and phase-separating proteins. The method exploits the density changes that occur when phase-separating proteins undergo oligomerization during condensate formation. In H1975 cells, we identified over 1,500 phase-separating proteins under osmotic compression or TGF-β treatment; 538 of these candidates were not present in PhaSepDB, a database that compiles in vivo, in vitro and omics-derived proteins. The approach detects constitutive condensates and proteins that dynamically phase-separate in response to osmotic stress or TGF-β signaling. This protocol provides proteome-wide analysis of fractions of proteins having different densities and enables temporal resolution of phase-separation events. The procedure takes ~9 d and requires expertise in cell culture, biochemistry and mass spectrometry. This method enables systematic study of biomolecular condensates and disease-associated phase-separation mechanisms.
Indexed as
Biomolecular CondensatesHigh-Throughput Screening AssaysProteinsProteomicsCentrifugation, Density GradientHumansMass SpectrometryOsmotic PressurePhase SeparationTransforming Growth Factor betaProteinsTransforming Growth Factor beta
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