Developing Mass Spectrometry-based Approaches to Characterize Mono- and Poly(ADP-ribosyl)ated Proteomes

项目摘要 翻译后修饰（PTM）涉及向蛋白质添加化学基团， 调节经修饰的蛋白质的结构或功能以协调细胞过程。 ADP-核糖基化，即腺苷二磷酸核糖加到蛋白质上，存在于两种细胞中， 单体（MAR化）和聚合物（PAR化）形式，允许更复杂的调节。 ADP-核糖基化形式的合成和降解之间的平衡是基础， 调节细胞过程。这种平衡的破坏导致人类疾病，如癌症， 神经变性然而，关于这两个组织各自的贡献， MARylation和PARylation对生理过程和疾病的调节 发病机制基于质谱（MS）的蛋白质组学方法是用于 研究健康和疾病状态下的PTM。以前发表的蛋白质组学方法 研究ADP-核糖基化已经成功地鉴定了氨基酸上的修饰位点 具有不同的化学性质。然而，所有这些方法都不能鉴定ADP-核糖基化 与每个站点相关联的表单。该提案旨在开发基于MS的蛋白质组学方法， 在蛋白质组水平上鉴定ADP-核糖基化的位点和形式。为了保存信息 关于ADP-核糖基化在修饰位点的形式，我的方法将（a）保持ADP-核糖基化， （B）确定所述氨基酸是否是MAR化的，或 PARylated.有了区分每个位点ADP核糖基化形式的工具，我们可以 鉴定健康人和哺乳动物中MAR化和PAR化蛋白质的潜在功能差异， 疾病状态。最终，这一工作流程将为生物功能提供有价值的见解， 不同形式的ADP-核糖基化在生理和疾病状态。

PROJECT SUMMARY Post-translational modifications (PTMs) involve the addition of chemical groups to proteins, modulating the structures or functions of the modified proteins to coordinate cellular processes. ADP-ribosylation, the addition of adenosine diphosphate ribose to proteins, exists in both a monomeric (MARylation) and polymeric (PARylation) form, allowing for more complex regulation. The balance between the synthesis and degradation of ADP-ribosylation forms is fundamental for regulating cellular processes. Disruption of this balance leads to human diseases like cancer and neurodegeneration. However, much remains to be learned about the respective contributions of MARylation and PARylation to the regulation of physiological processes and disease pathogenesis. Mass spectrometry (MS)-based proteomics methods are the go-to techniques for investigating PTMs in healthy and disease states. Previously published proteomics methods studying ADP-ribosylation have been successful in identifying modification sites on amino acids of varied chemical properties. However, all of these methods fail to identify the ADP-ribosylation form associated with each site. This proposal aims to develop a MS-based proteomics method to identify the site and form of ADP-ribosylation on a proteome-wide level. To preserve information about the form of ADP-ribosylation at modification sites, my method will either (a) keep ADP- ribosylation intact during enrichment or (b) determine whether the amino acid is MARylated or PARylated. With a tool to distinguish between the ADP-ribosylation forms at each site, we can identify potential functional differences of MARylated and PARylated proteins in healthy and disease states. Ultimately, this workflow will provide valuable insights into the biological functions of the different forms of ADP-ribosylation in physiological and disease states.