High Resolution and Multidimensional Protein Condensate Phase Diagrams with a Combinatorial Microdroplet Platform

The assembly of intracellular proteins into biomolecular condensates through phase separation is a fundamental process underlying organisation of the intracellular space and regulation of many central cellular processes. Elucidating the physico-chemical parameters that modulate phase separation is crucial in order to understand its effects in health and disease, as well as to develop therapeutic strategies that target biomolecular condensate systems. Phase behaviour is described by the phase diagram that defines conditions at which distinct phases exist, as well as the boundaries between these phases. Phase diagrams are typically built up from numerous individual measurements sampling different parts of the phase space. For biomolecular condensates, this process is slow, low throughput and associated with significant sample consumption even when performed in microwell format. Due to the rapidly increasing number of biologically and disease-relevant condensate systems, experimental techniques that enable high-throughput analysis of protein phase behaviour are required. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for the rapid and high-resolution acquisition of protein phase diagrams in multiple chemical and physical dimensions. Using this platform, we characterise the phase behaviour of a wide range of protein systems under a variety of conditions and show that this platform can be used to characterise quantitatively the effect of small molecules on biomolecular phase transitions.