Tandem lectin weak affinity chromatography for glycoprotein enrichment

In this chapter we describe the application of lectin weak affinity chromatography (LWAC) for the enrichment of peptides modified by O-linked β-N-acetylglucosamine (O-GlcNAc). O-GlcNAc is a single carbohydrate moiety post-translational modification of intracellular proteins. The stoichiometry of the modification is low and identification of the sites of O-GlcNAc attachment is challenging. To map O-GlcNAc sites we use the approach where a protein sample of interest is digested with trypsin and subjected to LWAC, which employs weak interaction between lectin wheat germ agglutinin and O-GlcNAc. Obtained sample is enriched with O-GlcNAc-modified peptides, which can be identified by means of mass spectrometry.