A rapid, high-throughput assay for the detection of O-GlcNAcylation

The level of O-GlcNAcylation is a critical sensor of cellular metabolic status. However, high-throughput quantification of protein O-GlcNAcylation remains a great challenge owing to its low abundance and labile nature. In this study, we developed a Quantitative Dot Blot (QDB)-based assay for quantifying the global O-GlcNAcylation of proteins in complex biological samples. For the detection of O-GlcNAcylation proteins, the QDB method exhibited high sensitivity, yielding a linear response over the range of 2.5-20 μg of total protein with a coefficient of determination (R²) of 0.99, and the limit of detection was determined to be 2.5 μg. And the QDB assay exhibited robust performance in quantifying variable O-GlcNAcylation levels in TMG-treated HEK293T cells, with a linear regression R² of 0.9353. We further validated the applicability of the QDB method in tissues from diabetic mice, including the soleus muscle and retina. The trends in O-GlcNAcylation changes detected by QDB were consistent with those obtained via Western blot (WB). Collectively, these data demonstrate that the QDB assay can be applied to measure O-GlcNAcylation levels in diverse tissue types. It may thus serve as an effective tool for evaluating O-GlcNAcylation activity and inhibitory effects in clinical diagnostics. Moreover, the QDB platform could potentially be extended to the detection and monitoring of other post-translational modifications.