Development of mass spectrometry methods to investigate conformations and complexes of proteins relating to health and disease.

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Liquid-liquid phase separation (LLPS) of protein molecules is an important process in normal cell physiology, and dysfunctional LLPS is linked with disease states including Alzheimer's disease and amyotrophic lateral sclerosis (ALS). Despite its importance, the molecular mechanisms underlying LLPS remain poorly understood due to a lack of methods suitable for tracking the conformations of proteins throughout the process. We have recently demonstrated that ion mobility mass spectrometry (IMMS) is an effective method for differentiating protein conformations in conditions that promote and reverse LLPS, and discovered that LLPS of the ubiquitin (Ub) shuttle protein Ubiquilin-2 (UBQLN2) is promoted via its elongation and inhibited by its compaction. The aim of this research is to use these novel IMMS tools to answer the following questions:(1) What are the fundamental LLPS mechanisms across the Ub shuttle proteins?(2) What are the differences in LLPS mechanisms in healthy and disease states?(3) How does LLPS of the Ub shuttle proteins contribute to proper cell homeostasis? This work will uncover mechanisms of LLPS in normal cell physiology, as well as inform on the alterations of the protein conformations in disease states. Mutations in the UBQLN family of shuttle proteins (UBQLN1/2/4) are associated with Alzheimer's and ALS, and altered shuttle protein expression level and mislocalisation are also associated with disease states, as increased amounts of shuttle factor Rad23 are detected in ALS. We speculate that disrupted protein homeostasis associated with shuttle protein condensates results in aggregates characteristic of neurodegenerative disorders, thus underscoring the need to determine the biophysical principles relating to LLPS.