SELECTIVE DETECTION AND SITE-ANALYSIS OF O-GLCNAC-MODIFIED GLYCOPEPTIDES BY BETA-ELIMINATION AND TANDEM ELECTROSPRAY MASS-SPECTROMETRY

Over the past decade, a number of nuclear and cytoplasmic proteins hav e been identified that are modified by single N-acetylglucosamine resi dues attached to the hydroxyl side chain of serines or threonines (O-G lcNAc). O-GlcNAc is a dynamic modification and therefore may act in a regulatory capacity analogous to phosphorylation. To undertake site-di rected mutagenesis studies of O-GlcNAc's function, it is necessary to identify the sites of glycosylation on various proteins. The current m ethod of site mapping, which involves galactosyltransferase labeling, generation of glycopeptides by proteolysis, purification by several ro unds of HPLC, and gas-phase and manual Edman sequencing, is very tedio us and requires about 10 pmol of pure, labeled glycopeptide. In this r eport, synthetic glycopeptides were generated and used to demonstrate that O-GlcNAc-modified peptides can be rapidly identified in complex m ixtures by HPLC-coupled electrospray mass spectrometry due to the part ial loss of the O-linked glycan (204 amu) at a modest orifice potentia l. Furthermore, the exact site of glycosylation was directly identifie d in the low picomole range by collision-induced dissociation (CID) of the glycopeptide after removal of the O-GlcNAc by alkaline beta-elimi nation. The conversion of glycosylserine to 2-aminopropenoic acid (2-a p) by beta-elimination both decreased the mass of the glycopeptide by 222 amu and resulted in a CID fragment ion representing the loss of 69 amu (2-ap) instead of 87 amu (Ser) at the position of the glycosylser ine. Finally, we tested this method on an identical synthetic, alpha-l inked O-GalNAc-modified peptide. Like O-GlcNAc, the O-GalNAc moiety wa s selectively removed at a modest orifice potential; however, the beta -elimination conditions that efficiently removed the O-GlcNAc only lib erated about 20% of the O-GalNAc. We conclude that the selectivity and the sensitivity of this method will make it a powerful tool for deter mining the sites of O-GlcNAc modification on proteins of low abundance such as transcription factors and oncogenes. (C) 1996 Academic Press, Inc.