Discovery of O-GlcNAcmodified proteins in published large-scale proteome data

The attachment of N-acetylglucosamine to serine or thre-onine residues (O-GlcNAc) is a post-translational modifica-tion on nuclear and cytoplasmic proteins with emerging roles in numerous cellular processes, such as signal trans-duction, transcription, and translation. It is further pre-sumed that O-GlcNAc can exhibit a site-specific, dynamic and possibly functional interplay with phosphorylation. O-GlcNAc proteins are commonly identified by tandem mass spectrometry following some form of biochemical enrich-ment. In the present study, we assessed if, and to which extent, O-GlcNAc-modified proteins can be discovered from existing large-scale proteome data sets. To this end, we conceived a straightforward O-GlcNAc identification strategy based on our recently developed Oscore software that automatically analyzes tandem mass spectra for the presence and intensity of O-GlcNAc diagnostic fragment ions. Using the Oscore, we discovered hundreds of O-GlcNAc peptides not initially identified in these studies, and most of which have not been described before. Merely re-searching this data extended the number of known O-GlcNAc proteins by almost 100 suggesting that this modi-fication exists even more widely than previously anticipated and the modification is often sufficiently abundant to be detected without enrichment. However, a comparison of O-GlcNAc and phospho-identifications from the very same data indicates that the O-GlcNAc modification is consider-ably less abundant than phosphorylation. The discovery of numerous doubly modified peptides (i.e. peptides with one or multiple O-GlcNAc or phosphate moieties), suggests thatO-GlcNAc and phosphorylation are not necessarily mu-tually exclusive, but can occur simultaneously at adjacen