■固有无序蛋白质(IDPs)代表缺乏二级或三级结构的蛋白质家族。国内流离失所者是互动网络的枢纽,参与液-液相分离过程,并驱动无蛋白质膜细胞器的形成。它们的展开结构使它们特别容易发生翻译后修饰(PTM),这些修饰起着关键的功能调节作用。
■我们讨论了从IDP富集方法(强酸提取和基于热的预分馏)开始研究IDPs磷酸化的不同分析方法,富集和映射磷酸肽/蛋白质的策略,和基于质谱的工具来研究IDPs的磷酸化依赖性构象改变(有限的蛋白水解,HDX,化学交联,共价标记,和离子迁移率)。
■对国内流离失所者及其PTM的兴趣日益浓厚,因为它们与几种疾病有关。可以充分利用可用于研究IDP及其磷酸依赖性构象改变的基于结构质谱的方法,利用内在障碍来促进IDP的纯化和合成生产。具有离子迁移设备和电子转移解离能力的质谱仪的扩散和实施可能是增加IDP生物学信息的关键因素。
Intrinsically disordered proteins (IDPs) represent a family of proteins that lack secondary or tertiary structure. IDPs are hubs in interaction networks, participate in liquid-liquid phase separation processes, and drive the formation of proteinaceous membrane-less organelles. Their unfolded structure makes them particularly prone to post-translational modifications (PTMs) that play key functional modulatory roles.
We discuss different analytical approaches to study phosphorylation of IDPs starting from methods for IDP enrichment (strong acid extractions and heat-based pre-fractionation), strategies to enrich and map phosphopeptides/proteins, and mass spectrometry-based tools to study the phosphorylation-dependent conformational alterations of IDPs (limited proteolysis, HDX, chemical cross-linking, covalent labeling, and ion mobility).
There is a growing interest in IDPs and their PTMs since they are involved in several diseases. The intrinsic disorder could be exploited to facilitate purification and synthetic production of IDPs taking full advantage of those structural mass-spectrometry-based methods that can be used to investigate IDPs and their phospho-dependent conformational alterations. The diffusion and implementation of mass spectrometers with ion mobility devices and electron transfer dissociation capabilities could be key-elements for increasing information on IDP biology.