Detection and identification of <i>O</i> -GlcNAc-modified proteins using 6-azido-6-deoxy- <i>N</i> -acetyl-galactosamine

Abstract

An unnatural monosaccharide with a C6-azide, Ac36AzGalNAc, has been developed as a potent and selective probe for O-GlcNAc-modified proteins. Combined with click chemistry, we demonstrate that Ac36AzGalNAc can robustly label O-GlcNAc glycosylation in a wide range of cell lines. Meanwhile, cell imaging and LC-MS/MS proteomics verify its selective activity on O-GlcNAc. More importantly, the protocol presented here provides a general methodology for tracking, capturing and identifying unnatural monosaccharide modified proteins in cells or cell lysates.