β-O-linkedN-acetylglucosamine (O-GlcNAc) is posttranslationally added to serine and threonine residues of many nuclear and cytoplasmic proteins found in metazoans. This modification is dynamic and responsive to numerous stimuli and conditions, suggesting an important role in many regulatory pathways. Moreover, the O-GlcNAc modification seems to compete with phosphorylation for sites of attachment, indicating a reciprocal relationship with phosphorylation. This chapter includes protocols for: (1) identifying the O-GlcNAc modification on proteins through immunoblotting, lectin affinity chromatography, and galactosyltransferase labeling; and (2) identifying and enriching for the sites of attachment using the mass spectrometry-based β -e limination followed by M ichael a ddition with d ithiothreitol (BEMAD) technique.
