A high-throughput assay for O-GlcNAc transferase detects primary sequence preferences in peptide substrates

O-GlcNAc transferase (OGT) catalyzes the addition of N-acetylglucosamine (O-GlcNAc) onto a diverse array of intracellular proteins. Although hundreds of proteins are known to be modified by O-GlcNAc, a strict amino acid consensus sequence for OGT has not been identified. In this study, we describe the development of a high-throughput assay for OGT and use it to profile the specificity of the enzyme among a panel of peptide substrates.