O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic, reversible monosaccharide modifier of serine and threonine residues on intracellular protein domains. Crosstalk between O-GlcNAcylation and phosphorylation has been hypothesized. Here, we identified over 1750 and 16,500 sites of O-GlcNAcylation and phosphorylation from murine synaptosomes, respectively. In total, 135 (7%) of all O-GlcNAcylation sites were also found to be sites of phosphorylation. Although many proteins were extensively phosphorylated and minimally O-GlcNAcylated, proteins found to be extensively O-GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the O-GlcNAcylation system is specifically targeting a subset of the proteome that is also phosphorylated. Both PTMs usually occur on disordered regions of protein structure, within which, the location of O-GlcNAcylation and phosphorylation is virtually random with respect to each other, suggesting that negative crosstalk at the structural level is not a common phenomenon. As a class, protein kinases are found to be more extensively O-GlcNAcylated than proteins in general, indicating the potential for crosstalk of phosphorylation with O

O‐linked N‐acetylglucosamine (O‐GlcNAc) is a dynamic, reversible monosaccharide modifier of serine‐ and threonine‐residues on intracellular protein domains. Crosstalk between O‐GlcNAc and phosphorylation signaling mechanisms has been proposed. However, to date there has not been a large scale evaluation of these two PTMs at endogenous levels in any complex biological system. Here, we identified over 1,750 GlcNAcylation and16,500 phosphorylation sites from the same mouse brain synaptosomes.As has previously been demonstrated for phosphorylation, we show that GlcNAcylation tends to occur in clusters on disordered regions of proteins. However, the distribution of GlcNAcylation sites is virtually random with respect to phosphorylation, suggesting that negative crosstalk at the structural level is not a general phenomenon. We frequently observed proteins extensively phosphorylated with minimal GlcNAcylation. However, proteins extensively GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the process of GlcNAcylation appears functionally correlated to phosphorylation.