A Chemoproteomic Approach for System-Wide and Site-Specific Uncovering of Functional Protein N-Glycosylation

作者：Guoli Wang, Shiyun Ma, Hao Song, Yuying Liang, Xinze Li, Lei Zhang, Haojie Lu, Ying Zhang · 发表于：Journal of the American Chemical Society · 年份：2025 · DOI：10.1021/jacs.5c08065 · 被引用次数：8 · 研究领域：Glycosylation and Glycoproteins Research、RNA and protein synthesis mechanisms、Galectins and Cancer Biology

Technological advances in proteomics, including sample separation, mass spectrometry, and searching algorithms, have empowered in-depth discovery of protein post-translational modifications from biological samples. However, there is still a considerable delay in systematic research on the functional significance of these modifications. Herein, we develop a new thermal proteomic strategy, Refined-TPP, enabling the efficient study of protein thermostability with improved sensitivity and increased throughput by 5-fold. Its robust performance in target identification was demonstrated by the metabolite NADPH as well as the drug panobinostat. We further propose glyco-dependent thermal shift profiling (GTSP), a novel chemical proteomic methodology, to systematically examine the effects of site-specific modifications on the thermal stability of native proteins. Finally, we probed 208 functionally important N-glycosites mapping 113 proteins and elucidated their pivotal roles in protein functions, including protein stability, subcellular localization, and enzyme activity. This globally biophysical assay bridges the gap between structural modifications and their functional impacts on proteins and provides here a robust platform for the first time to effectively interrogate functional implications of N-glycosylation. It is also readily applicable in a high-throughput and unbiased manner to further investigations into diverse objects, including drug-target screening, protein interactions,...