High-throughput experimental methods for investigating biomolecular condensates

Abstract

Background: The concept of biomolecular condensate was put forward recently to emphasize the ability of certain cellular compartments to concentrate molecules and comprise proteins and nucleic acids with specific biological functions, from ribosome genesis to RNA splicing. Due to their unique role in biological processes, it is crucial to investigate their compositions, which is a primary determinant of condensate properties.

Results: Since a wide range of macromolecules comprise biomolecular condensates, it is necessary for researchers to investigate them using high-throughput methodologies while low-throughput experiments are not efficient enough. These high-throughput methods usually purify interacting protein libraries from condensates before being scanned in mass spectrometry. It is possible to extract organelles as a whole for specific condensates for further analysis, however, most condensates do not have a distinguishable marker or are sensitive to shear force to be extracted as a whole. Affinity tagging allows a comprehensive view of interacting proteins of target molecule yet only proteins with strong bonds may be pulled down. Proximity labeling serves as a complementary method to label more dynamic proteins with weaker interactions, increasing sensitivity while decreasing specificity. Image-based fluorescent screening takes another path by scanning images automatically to illustrate the condensing state of biomolecules within membraneless organelles, which is a unique feature unlike the previous mass spectrometry-based methods.

Conclusion: This review presents a rough glimpse into high-throughput methodologies for biomolecular condensate investigation to encourage usage of bioinformatic tools by researchers in relevant fields.

Author summary

Biomacromolecules often form condensates in cytoplasm or nucleoplasm to keep biomolecular reactions organized and strictly regulated. The past 10 years have witnessed a surge in researches concerning biomolecular condensates and phase-separating molecules, yet most experiments are performed in low-throughput manners. This review summarizes the common high-throughput experimental methods for biomolecular condensate investigation, including organelle purification, affinity purification, proximity labeling, and image-based fluorescence screening and discusses their advantages and shortcomings. We demonstrate that high-throughput screening may provide new insights into related studies and encourage researchers to develop new tools to unravel phase-separated biomolecular condensates.

Graphical abstract

Keywords

biomolecular condensates / high-throughput / phase separation / interaction

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ACKNOWLEDGEMENTS

This work was supported by the National Key Research and Development Program of China (2018YFA0507504), the National Natural Science Foundation of China (61773025, 32070666), Clinical Medicine Plus X - Young Scholars Project of Peking University (PKU2021LCXQ012) and the Fundamental Research Funds for the Central Universities.

COMPLIANCE WITH ETHICS GUIDELINES

The authors Taoyu Chen, Qi Lei, Minglei Shi and Tingting Li declare that they have no conflict of interests.

This article is a review article and does not contain any studies with human or animal subjects performed by any of the authors.

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