Identification and Characterization of GRASP55 O-GlcNAcylation.

O-GlcNAcylation is a posttranslational modification of proteins that adds a single sugar, β-N-acetylglucosamine (GlcNAc), to Ser/Thr residues. Extensive studies have been conducted to identify and characterize substrates of this modification but mostly focused on cytosolic and nuclear proteins. This chapter describes a detailed protocol used to determine the O-GlcNAcylation of GRASP55, the first O-GlcNAcylated Golgi protein identified, including how its O-GlcNAcylation level responds to glucose deprivation. In addition, this chapter also provides a detailed method to express and purify O-GlcNAcylated GRASP55 in bacteria for further in vitro functional assays. This protocol could be applied to other Golgi proteins that are potentially O-GlcNAcylated.