| O-linked-beta-N-acetylglucosamine (O-GlcNAc) is a post-translational modification to serines and threonines of nuclear and cytoplasmic proteins. Since its discovery, GlcNAcylation has been found to have an abundance and distribution similar to phosphorylation. However, while thousands of phosphorylation sites have been identified, <200 O-GlcNAc sites have been characterized due to enormous analytical challenges. Mass spectrometric techniques have, until recently, limited the study of O-GlcNAc to the identification of modified source proteins. O-linked GlcNAc is highly labile and is lost from the peptide backbone upon conventional collision-activated dissociation (CAD), making unambiguous site identification nearly impossible. The following dissertation presents several research projects demonstrating the utility of electron-transfer dissociation (ETD) mass spectrometry for studying the O-GlcNAc modification. ETD is soft dissociation technique relative to CAD, and as a result, fragmentation of peptide ions by ETD leaves labile modifications intact. Using ETD, we successfully characterized O-GlcNAc sites on two proteins, FoxO1 and PGC-1alpha, involved in transcriptional regulation of gluconeogenesis. The O-GlcNAc site identified on Thr317 of FoxO1 was to found play a role in FoxO1 activation in response to glucose, resulting in the paradoxical increase in expression of gluconeogenic genes.;While ETD overcomes the limitations of conventional mass spectrometric methods, characterization of low-level O-GlcNAc sites is challenging because existing enrichment methods often proceed in poor yield. Additionally, modified residues often occur in regions of proteins containing clusters of serines and threonines and a paucity of basic residues. To overcome these challenges, we developed a photocleavable-biotin-alkyne tag that facilitates enrichment of O-GlcNAc peptides from complex protein mixtures. This tag was specifically designed for ETD as it adds a basic group to the modification site and guarantees that tryptic peptides will carry at least three charges and provide high-quality sequence information. A quantitative proteomics experiment for studying O-GlcNAc dynamics during M phase is also presented. In this study, the combination of differential labeling with this tagging/enrichment strategy enabled the identification of the largest set of O-GlcNAc sites to date. In addition, phosphopeptides were also enriched and quantified, and the combined data enabled evaluation of the interplay between phosphorylation and GlcNAcylation on regulatory and cytoskeletal proteins associated with mitosis. |
