Analytical Methods for the Study of O-GlcNAc Glycoproteins and Glycopeptides

Over the past decade, numerous nuclear and cytosolic glycoproteins have been shown to be modified by single N -acetylglucosamine (O -GlcNAc) residues attached to the side chain hydroxyl groups of serine or threonine (1 –3 ). This unique postranslational modification (O -GlcNAcylation), described in lymphocytes by Torres and Hart (4 ), can be found on a wide range of proteins including cytoskeletal proteins (5 –8 ) nuclear pore proteins (9 –13 ), transcription factors (41 ), RNA polymerase II (15 ), oncoproteins (16 ,17 ), and viral proteins (18 –20 ), to name a few. In addition to the O -GlcNAc modification, all of these proteins are known phosphoproteins and many have been reported to be regulated and/or form reversible protein complexes based on their state of phosphorylation. These observations suggest that O -GlcNAcylation also plays a role in the assembly/disassembly of protein complexes and/or the regulation of enzymes, perhaps by acting as an antagonist to phosphorylation. In fact, several studies have shown that phosphorylation and O -GlcNAcylation can occur at the same sites on a given protein (15 ,21 ). For example, a recent study documents that the c-Myc oncoprotein is O -GlcNAcylated at threonine 58, a known site of phosphorylation and mutational hotspot for c-Myc in human Burkitt and AIDS-related lymphomas (21 ). Furthermore, O -GlcNAcylation appears to be a key regulatory modification since it is highly dynamic (22 ,23 ), responsive to external cellular stimuli (24 ), and regulated in a cell cycle-dependent manner (25 ). However, direct evidence for the function of O -GlcNAcylation has been limited, in large part, because of the difficulty and the amount of protein required for mapping sites of glycosylation as a prerequisite to molecular manipulations such as site-directed mutagenesis. The primary method for mapping sites of O -GlcNAcylation requires the enzymatic transfer of a [³ H]galactose with bovine milk galactosyltransferase to tag the O -GlcNAc-modified protein, generation of glycopeptides by proteolysis, purification of the glycopeptides by several rounds of high-performance liquid chromatography (HPLC), followed by gas-phase and manual Edman sequencing. Each of these steps is very tedious and often results in significant losses in yield as a result of incomplete proteolysis, poor resolution of the glycopeptides by HPLC, and conservative pooling of HPLC fractions to maximize purity of the glycopeptides. Finally, this method requires a minimum of about 10 pmol of pure, labeled, glycopeptide to generate unambiguous sequence and to identify the site of glycosylation. Therefore, methods to improve recoveries of glycopeptides and increase the sensitivity of the site analysis assays would be a major breakthrough in the study of O -GlcNAcylation.