Milestones in O-GlcNAc Research: Technological Advances that Shaped the Field.

Since its discovery over forty-years ago, newly developed and improved existing technologies have played a crucial role in advancing O-GlcNAcylation research. We now know that O-GlcNAcylation is central to nutrient/stress regulation of nearly all cellular processes. O-GlcNAcylation was discovered by using labeling by galactosyltransferase and tritiated UDP-GlcNAc combined with product analyses. This enzymatic probe remained the gold standard for early studies in the field. Subsequent development of O-GlcNAc specific antibodies greatly advanced the field by providing non-glycobiologists facile methods for identification of O-GlcNAcylated proteins. Purification and cloning of O-GlcNAc cycling enzymes allowed for development of specific inhibitors and for genetic studies to elucidate the functions of the sugar. Since O-GlcNAc is so labile in the gas phase and is subject to severe ion-suppression in classical collision-induced fragmentation, the development of electron transfer dissociation mass spectrometry was a major advance in O-GlcNAc glycoproteomics, leading to the development of large databases of O-GlcNAcylated proteins. Bioorthogonal chemistry provided valuable new tools to evaluate O-GlcNAc functions in living cells. These and other technological advances have played a major role during the past four decades in establishing O-GlcNAcylation as a major regulator of cellular metabolism.