Generation of O-GlcNAc specific monoclonal antibodies using a novel synthetic immunogen

O-GlcNAc modification is a ubiquitous and dynamic post-translational modification that has been implicated in many cellular processes, frequently via interplay with phosphorylation that can occur on the same serine and threonine residues. Unlike phosphorylation for which a wide range of pan- and site-specific phospho-antibodies are available, only two pan-O-GlcNAc specific antibodies have been described: an IgM pan-O-GlcNAc antibody and an IgG antibody raised against O-GlcNAc modified components of the nuclear pore complex. In fact, it is well known that O-GlcNAc modified glycoconjugates do not elicit relevant IgG isotype antibodies. Here, we report a novel method combining a fully synthetic three-component immunogen with classical hybridoma technology to obtain a series of O-GlcNAc-specific IgG monoclonal antibodies. Large-scale shotgun proteomics approach utilizing these antibodies led to the identification of more than 200 O-GlcNAc modified proteins, of which approximately two-thirds were novel. Application of this tool allowed us to delineate differentially modified proteins of the liver in response to hemorrhagic trauma and resuscitation in a rat model.