Identifying Novel Phase Separation Proteins in E. coli and Human Cells using SEC-MS

Abstract

Phase separation of proteins and RNAs within cells has emerged as an important mechanism incellular organization and regulation of metabolism. Many facets of RNA biology take place in phase separated assemblies. Examples include RNA decay in mammalian processing bodies or bacterial RNP-bodies and ribosome biogenesis in the nucleolus of eukaryotic cells. Phase separation is driven by assembly of proteins and nucleic acids, each with numerous residues capable of promiscuous interaction, resulting in a highly heterogenous multivalent assembly. Methodologies to study these multivalent assemblies have relied heavily upon microscopy, and new approaches are needed to further characterize these assemblies. We have developed a method using size exclusion chromatography (SEC) and mass spectrometry to study multivalent, phase separated assemblies. Due to the weak nature of interactions comprising these assemblies, we employ formaldehyde crosslinking to stabilize them prior to cell lysis and SEC fractionation. Without crosslinking, many multivalent assemblies disassemble upon cell lysis. After lysates are separated according to particle size, we detect the protein constituents of the large protein particles with mass spectrometry. With this approach we have identified transcription granules containing RNA Pol II and RNA binding protein FUS. Also identified are novel protein constituents of multivalent phase separated protein assemblies in E. coli to expand upon existing literature. Additionally, we have identified proteins differentially integrated into multivalent protein assemblies in Ewing sarcoma cells.