翻译后修饰 (PTM) ADP-核糖基化在细胞信号传导和调节蛋白质功能中发挥着重要作用，并且与多种疾病的发展有关，包括乳腺癌和卵巢癌。然而，由于缺乏适当的工具来可靠地鉴定活细胞环境中生理相关的 ADP-核糖基化蛋白，因此研究这种 PTM 促进疾病发展的潜在机制受到了阻碍。在此，我们探索了炔烃标记探针 6Yn-ProTide-Ad (6Yn-Pro) 作为化学工具的应用，用于通过代谢标记鉴定细胞内 ADP 核糖基化蛋白质。我们在用 6Yn-Pro 处理的 HEK293T 细胞中应用靶向代谢组学和化学蛋白质组学，以证明探针在细胞内代谢转化为 ADP-核糖基化辅因子 6Yn-NAD ，并随后在氢气下标记和富集细胞中的 PARP1 和多种已知的 ADP-核糖基化蛋白过氧化物引起的应激。我们预计此处描述的方法和方法将有助于未来鉴定新型细胞内 ADP 核糖基化蛋白。本期刊版权所有 © 英国皇家化学学会。

The post-translational modification (PTM) ADP-ribosylation plays an important role in cell signalling and regulating protein function and has been implicated in the development of multiple diseases, including breast and ovarian cancers. Studying the underlying mechanisms through which this PTM contributes towards disease development, however, has been hampered by the lack of appropriate tools for reliable identification of physiologically relevant ADP-ribosylated proteins in a live-cell environment. Herein, we explore the application of an alkyne-tagged proprobe, 6Yn-ProTide-Ad (6Yn-Pro) as a chemical tool for the identification of intracellular ADP-ribosylated proteins through metabolic labelling. We applied targeted metabolomics and chemical proteomics in HEK293T cells treated with 6Yn-Pro to demonstrate intracellular metabolic conversion of the probe into ADP-ribosylation cofactor 6Yn-NAD+, and subsequent labelling and enrichment of PARP1 and multiple known ADP-ribosylated proteins in cells under hydrogen peroxide-induced stress. We anticipate that the approach and methodology described here will be useful for future identification of novel intracellular ADP-ribosylated proteins.This journal is © The Royal Society of Chemistry.