A Multi-Method Approach to Analyzing O-GlcNAc Modified Peptides

Abstract

The O-linked β-N-acetyl glucosamine (O-GlcNAc) modification, a dynamic post-translational modification (PTM) to serine or threonine residues of nuclear and cytoplasmic proteins from almost all functional classes, is involved in many different cellular processes including signal transduction, protein degradation, and regulation of gene expression. O-GlcNAc modification is known to be associated with several human disease states, such as diabetes, cancer, cardiovascular and neurodegenerative disorders. Despite the vital functional roles of protein O-GlcNAcylation in many cellular processes, the area of O-GlcNAc research field has been hampered, mainly due to the lack of techniques for the identification, quantification and site mapping of O-GlcNAc modification in proteins. Proteomic analysis of O-GlcNAc modified proteins still presents significant challenges following reasons. First, site mapping of O-GlcNAcylation is very difficult due to its collision-induced dissociation (CID)-labile β-linkage between O-GlcNAc moiety and serine or threonine residues. Second, O-GlcNAc modified proteins are present in low stoichiometry at each site on proteins. Therefore, the research of O-GlcNAc modification has been limited by difficulties in mapping sites of O-GlcNAc modification. In this study, a new multi-method approach is introduced using enrichment and novel N-terminus tag based on the application of CID tandem mass spectrometry for O-GlcNAc proteome profiling. To compensate the substoichiometric occupancy and the lability of O-GlcNAc modified proteins, a new method for enrichment and detection is developed. The approach is combined with (i) lectin weak affinity chromatography (LWAC) and (ii) TEMPO [2-(2,2,6,6-tetramethyl piperidine-1-oxyl)]-assisted free radical-initiated peptide sequencing mass spectrometry (FRIPS MS). Collectively, with combined with LWAC and TEMPO-assisted FRIPS MS, this approach can be used in a variety of applications for O-GlcNAc research, all of which will provide insights into the many functions of O-GlcNAcylation in biological systems. The approach is not only improving enrichment for O-GlcNAc modified peptides but also allowing confirmation of a number of O-GlcNAcylation sites. We envisage that further application of the method to biological systems, which will elucidate the mechanism of specific role of protein O-GlcNAcylation in many biological processes. The new multi-method provides an ideal platform to profile global O-GlcNAc peptides for quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and will enable the study of functional roles of O-GlcNAc modified peptide in biological systems.