Quantitative Profiling of Protein O‑GlcNAcylation Sites by an Isotope-Tagged Cleavable Linker

• Published in: ACS chemical biology Vol. 13; no. 8; pp. 1983 - 1989
• Main Authors: Qin, Ke, Zhu, Yuntao, Qin, Wei, Gao, Jinjun, Shao, Xuan, Wang, Yan-ling, Zhou, Wen, Wang, Chu, Chen, Xing
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.08.2018
• ISSN: 1554-8929; 1554-8937; 1554-8937
• DOI: 10.1021/acschembio.8b00414

Large-scale quantification of protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification in a site-specific manner remains a key challenge in studying O-GlcNAc biology. Herein, we developed an isotope-tagged cleavable linker (isoTCL) strategy, which enabled isotopic labeling of O-GlcNAc through bioorthogonal conjugation of affinity tags. We demonstrated the application of the isoTCL in mapping and quantification of O-GlcNAcylation sites in HeLa cells. Furthermore, we investigated the O-GlcNAcylation sensitivity to the sugar donor by quantifying the levels of modification under different concentrations of the O-GlcNAc labeling probe in a site-specific manner. In addition, we applied isoTCL to compare the O-GlcNAcylation stoichiometry levels of more than 100 modification sites between placenta samples from male and female mice and confirmed site-specifically that female placenta has a higher O-GlcNAcylation than its male counterpart. The isoTCL platform provides a powerful tool for quantitative profiling of O-GlcNAc modification.