[26] Detection of O-Linked N-acetylglucosamine (O-GlcNAc) on cytoplasmic and nuclear proteins

This chapter describes methods for the detection and initial analysis of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. Although the galactosyltransferase/UDP[3H]galactose probe method is highly specific it requires amounts of protein in the low microgram range. The chapter also describes a coupled transcription/translation/lectin chromatography method for the detection of O-GIcNAc on low-abundance proteins for which a cDNA is available. This method relies on the observation that the reticulocyte lysates commonly used for in vitro translation already contain sufficient sugar nucleotide and O-GIcNAc transferases to glycosylate the translation products efficiently. Because O-GIcNAc is virtually the only GlcNAc-containing molecule in the cytoplasmic or nuclear cellular compartments, metabolic radiolabeling with [3H]glucosamine, in conjunction with subcellular fractionation, is also a useful method for identifying O-GlcNAc-bearing proteins. Galactosyltransferase is a specific and sensitive probe frequently used in the detection of O-GlcNAc on cytoplasmic and nuclear proteins. The radiolabeled products are then analyzed to determine saccharide linkage (O- or N-linkage) and structure. In some cases, this method is sensitive enough to detect O-GIcNAc in the subpicomole range.