An Isotope-Coded Photocleavable Probe for Quantitative Profiling of Protein O‑GlcNAcylation

O-linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification of proteins and is essential for cell function. Quantifying the dynamics of O-GlcNAcylation in a proteome-wide level is critical for uncovering cellular mechanisms and functional roles of O-GlcNAcylation in cells. Here, we develop an isotope-coded photocleavable probe for profiling protein O-GlcNAcylation dynamics using quantitative mass spectrometry-based proteomics. This probe enables selective tagging and isotopic labeling of O-GlcNAcylated proteins in one step from complex cellular mixtures. We demonstrate the application of the probe to quantitatively profile O-GlcNAcylation sites in 293T cells upon chemical induction of O-GlcNAc levels. We further applied the probe to quantitatively analyze the stoichiometry of O-GlcNAcylation between sorafenib-sensitive and sorafenib-resistant liver cancer cells, which lays the foundation for mechanistic investigation of O-GlcNAcylation in regulating cancer chemoresistance. Thus, this probe provides a powerful tool to profile O-GlcNAcylation dynamics in cells.O-连接N-乙酰葡糖胺（O-linked N-acetylglucosamine, O-GlcNAc）是一种普遍存在的蛋白质翻译后修饰，对细胞功能不可或缺。在蛋白质组全局层面定量解析O-GlcNAc糖基化的动态变化，对于揭示细胞内O-GlcNAc糖基化的调控机制与功能作用具有关键意义。本研究开发了一款同位素编码光裂解探针，可结合基于定量质谱的蛋白质组学技术，实现对蛋白质O-GlcNAc糖基化动态的谱图分析。该探针可一步完成复杂细胞混合体系中O-GlcNAc糖基化蛋白的选择性标记与同位素标记。我们验证了该探针在化学诱导293T细胞O-GlcNAc水平后，对其O-GlcNAc糖基化位点进行定量谱图分析的应用效果。进一步，我们将该探针用于定量分析索拉非尼（sorafenib）敏感型与索拉非尼耐药型肝癌细胞间的O-GlcNAc糖基化化学计量比，为探究O-GlcNAc糖基化调控肿瘤化疗耐药性的分子机制奠定了基础。综上，该探针为研究细胞内O-GlcNAc糖基化动态提供了一款强有力的工具。