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

蛋白质分子的液-液相分离（LLPS）是正常细胞生理的一个重要过程，而功能失调的LLPS与阿尔茨海默病和肌萎缩侧索硬化症（ALS）等疾病状态有关。尽管它很重要，但由于缺乏在整个过程中跟踪蛋白质构象的合适方法，LLPS的分子机制仍然知之甚少。我们最近证明了离子迁移质谱（IMMS）是一种在促进和逆转LLPS条件下区分蛋白质构象的有效方法，并发现泛素（Ub）穿梭蛋白Ubiquilin-2 （UBQLN2）的LLPS通过其延伸而被促进，并通过其压实而被抑制。本研究的目的是利用这些新颖的IMMS工具来回答以下问题：(1)跨Ub穿梭蛋白的基本LLPS机制是什么？(2)健康状态和疾病状态下LLPS的机制有何差异？(3) Ub穿梭蛋白的LLPS如何促进适当的细胞稳态？这项工作将揭示LLPS在正常细胞生理学中的机制，以及疾病状态下蛋白质构象的改变。穿梭蛋白UBQLN家族的突变（UBQLN1/2/4）与阿尔茨海默病和ALS相关，穿梭蛋白表达水平的改变和错定位也与疾病状态相关，因为在ALS中检测到穿梭因子Rad23的量增加。我们推测，与穿梭蛋白凝聚体相关的蛋白质稳态破坏导致神经退行性疾病的聚集特征，因此强调需要确定与LLPS相关的生物物理原理。

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.