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Glyco-seek: Ultrasensitive Detection of Protein-Specific Glycosylation by Proximity Ligation Polymerase Chain Reaction

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journal contribution
posted on 25.07.2016, 00:00 by Peter V. Robinson, Cheng-ting Tsai, Amber E. de Groot, Julia L. McKechnie, Carolyn R. Bertozzi
We report a non-destructive biochemical technique, termed “Glyco-seek”, for analysis of O-GlcN­Acylated proteins. Glyco-seek combines chemoenzymatic labeling, proximity ligation, and quantitative polymerase chain reaction to detect O-GlcN­Acylated proteins with ultrahigh sensitivity. Our glycan-specific assay can be paired with traditional proximity ligation assays to simultaneously determine the change in total protein levels. We show that Glyco-seek detects attomoles of glycoproteins of interest from cell lysates, with sensitivity several orders of magnitude higher than that of current techniques. We used the method to directly assay the O-GlcN­Acylation status of a low-abundance transcription factor from cell lysates without need for isolation or enrichment.