A high-throughput method for exploring the parameter space of protein liquid-liquid phase separation

Numerous proteins can undergo liquid-liquid phase separation (LLPS) in various biological processes. However, it remains a challenge to systematically evaluate the LLPS ability of proteins. Here, we develop a high-throughput protein phase separation (HiPPS) profiling method to delineate LLPS ability of proteins, which we term as the LLPS space of proteins. We next apply HiPPS to explore LLPS space of more than 30 different proteins including RNA-binding proteins, chaperones, amyloid proteins, and enzymes. Our results demonstrate that (1) LLPS is a common property of both unstructured and well-folded proteins; (2) LLPS space of protein can be dynamically reshaped by protein-protein interaction, post-translational modification, and genetic mutation; and (3) multi-component co-LLPS mixture decreases the threshold concentration of individual proteins for LLPS. We anticipate that the HiPPS method may effectively characterize LLPS of individual proteins or protein mixtures in vitro, which is valuable for understanding its role in biological processes.