MOE Laboratory of Biosystems Homeostasis & Protection, College of Life Sciences , Zhejiang University , Hangzhou 310058 , China.
State Key Laboratory of Toxicology and Medical Countermeasures , Beijing Institute of Pharmacology and Toxicology , Beijing 100850 , China.
ACS Chem Biol. 2019 Jan 18;14(1):4-10. doi: 10.1021/acschembio.8b01052. Epub 2019 Jan 8.
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-连接的 N-乙酰葡萄糖胺（O-GlcNAc）是蛋白质上普遍存在的一种翻译后修饰，对细胞功能至关重要。在全蛋白质组水平上定量研究 O-GlcNAc 化的动态变化对于揭示细胞中 O-GlcNAc 化的细胞机制和功能作用至关重要。在这里，我们开发了一种同位素编码的光裂解探针，用于通过基于定量质谱的蛋白质组学来分析蛋白质 O-GlcNAc 化动力学。该探针能够在一步中从复杂的细胞混合物中选择性标记和同位素标记 O-GlcNAc 化蛋白。我们证明了该探针可用于在化学诱导 O-GlcNAc 水平升高时定量分析 293T 细胞中的 O-GlcNAc 化位点。我们进一步应用该探针定量分析索拉非尼敏感和耐药肝癌细胞之间的 O-GlcNAc 化的化学计量比，为研究 O-GlcNAc 化在调节癌症化疗耐药性中的机制奠定了基础。因此，该探针为研究细胞中 O-GlcNAc 化的动态变化提供了一种强大的工具。
