Ultrasensitive assay of O-GlcNAc transferase using capillary electrophoresis-laser induced fluorescence.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is directly associated with the level of O-GlcNAc glycosylation of biomolecules and various diseases, and it is expected to be a promising potential new therapeutic target. Here, we develop a robust and sensitive method for OGT assay based on capillary electrophoresis-laser induced fluorescence (CE-LIF) method. AF-488-modified peptide containing serine active group is designed as substrate for OGT-catalyzed reaction, and nonradioactive UDP-GlcNAc is employed as sugar donor to perform O-GlcNAc glycosylation modification. The enzyme activity of OGT is measured by quantitative determination of glycosylated peptide produced by the reaction. Large volume sample stacking technique for sample injection and a unique fluorescence collection system for LIF detection are adopted to greatly enhance the detection sensitivity, thus a low limit of detection down to 0.23 pM for OGT detection is achieved. The method is successfully applied to detect OGT activity in clinical blood samples with satisfactory accuracy. Our study provides a simple, accurate, and sensitive method with great potential application in clinical diagnosis of O-GlcNAc-related diseases.