Abstract
O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a dynamic protein posttranslational modification with roles in processes such as transcription, cell cycle regulation, and metabolism. Detailed mechanistic studies of O-GlcNAc have been hindered by a lack of methods for measuring O-GlcNAc stoichiometries and the interplay of glycosylation with other posttranslational modifications. We recently developed a method for labeling O-GlcNAc-modified proteins with resolvable poly(ethylene glycol) mass tags. This mass-tagging approach enables the direct measurement of glycosylation stoichiometries and the visualization of distinct O-GlcNAc-modified subpopulations. Here, we describe procedures for labeling O-GlcNAc glycoproteins in cell lysates with mass tags.