Novel Methods For Screening And Analysis Of Biomolecular Condensate Proteins

Liquid-liquid phase separation has proven to be a universal mechanism for the formation of biomolecular condensates in cells,which can regulate a variety of biological processes,such as gene expression and signal transduction.Researchers from the fields of biomedical engineering,cell biology,biophysics and structural biology are working to explore the molecular principles of this emerging field.For biomedical field,biomolecular condensates are considered as potential therapeutic targets for cancer and neurodegenerative diseases.In the field of engineering application,biomolecular condensates are used to design and synthesize new materials,such as fibrous materials and 3D printing materials.Although the research on the principle and application of biomolecular condensates is in full swing,high throughput screening methods for endogenous phase-separated proteins are still lacking.At the same time,high-throughput techniques are urgently needed to determine the phase separation behavior database of phase-separated proteins that are constantly being discovered.In addition,the biological function of phase-separated proteins is still not very clear,which restricts the further study of biomolecular condensates.To solve the above problems,this thesis establishes a high-throughput screening and analysis research method for protein condensates,and its main research contents are as follows:(1)A novel strategy for high-throughput screening and identification of endogenous phase-separated proteins was proposed by combining sucrose density gradient centrifugation with quantitative mass spectrometry.Volumetric compression was used to increase the concentration and crowding degree of proteins in cells and change the degree of protein oligomerization.Proteins with different oligomerization states are classified using sucrose density gradient centrifugation.Then,the proteins with significant changes in oligomerization state were identified and screened by quantitative mass spectrometry,and endogenous phase-separated proteins were screened at the proteomic level.Using this method,1 215 candidate phase-separated proteins were were successfully screened.Of these,462 newly discovered phase-separated proteins have never been reported.Candidate phase-separated proteins involved 11 known membraneless organelles(stress granules,etc.)proteome.Confocal imaging was used to verify the phase separation behavior of the protein in the cell,which proved the accuracy of the method.A larger dataset of endogenous phaseseparated proteins was obtained,which provided a method reference for the screening and identification of endogenous phase-separated proteins.(2)Based on the technique of centrifugal microfluidic,a rapid,high-throughput method for the determination of biomolecular condensates phase diagram was developed.Given the increasing identification of phase-separated proteins,there is an urgent need for high-throughput strategies to determine the phase separation behavior database of condensates.A centrifugal microfluidic gradient generator was designed,the sample was driven by capillary action to fill the microchannel automatically,the loading volume of samples was controlled by setting stop valves on the metering channel,and the samples were mixed under centrifugal force.With the in-situ evaporation of the samples,up to 1 750 concentration conditions could be generated within 140 minutes.The phase diagram of poly U/RRASLRRASLRRASL condensate was rapidly determined by this technique.The detection limit of critical saturation concentration of phase separation is about 5 times lower than that of traditional turbidity method.It provides a high-throughput and high-resolution biomolecular condensates research platform for biologists and chemists,and provides technical support for rapid and high-throughput acquisition of condensate phase diagrams.(3)The proteins that undergo phase separation during the biological process induced by TGF-β were screened and functionally analyzed.After the establishment of highthroughput screening method for endogenous phase-separated proteins and the determination technology of biomolecular condensate phase diagram,the phase-separated proteins that specifically respond to two typical biological processes of TGF-β signaling activation and TGF-β induced EMT were screened.A total of 73 phase-separated proteins were identified in response to TGF-β signaling activation and 509 cell EMT-related phaseseparated proteins were obtained,respectively.The biological functions of phase-separated proteins were analyzed.Besides the known biological functions,some unreported biological processes involved in the formation of condensates were also revealed,which provided a new idea for the research of the biological functions of phase-separated proteins.In summary,a high-throughput method for screening and identification of endogenous phase-separated proteins was established in this thesis;In order to obtain the database of the phase separation behavior of phase-separated proteins,a high-throughput technique for measuring the phase diagram of biomolecular condensates was developed;and finally,the biological function of phase-separated proteins in the biological process induced by TGF-βwas analyzed,which provided reference and inspiration for the screening,phase separation behavior analysis and biological function research of phase-separated proteins.