Discovery of O-GlcNAc-modified Proteins in Published Large-scale Proteome Data

Abstract

The attachment of N-acetylglucosamine to serine or threonine residues (O-GlcNAc) is a post-translational modification on nuclear and cytoplasmic proteins with emerging roles in numerous cellular processes, such as signal transduction, transcription, and translation. It is further presumed that O-GlcNAc can exhibit a site-specific, dynamic and possibly functional interplay with phosphorylation. OGlcNAc proteins are commonly identified by tandem mass spectrometry following some form of biochemical enrichment. 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 OGlcNAc 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 OGlcNAc proteins by almost 100 suggesting that this modification 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 considerably less abundant than phosphorylation. The discovery of numerous doubly modified peptides (i.e. peptides with one or multiple O-GlcNAc or phosphate moieties), suggests that O-GlcNAc and phosphorylation are not necessarily mutually exclusive, but can occur simultaneously at adjacent sites. Molecular & Cellular

Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation

“…We next queried whether these 1,286 protein sequences might not only be substrates of kinases and OGT but also might display interplay in between phosphorylation and O-GlcNAcylation, whereby phosphorylation at P−3 in this consensus sequence should prevent O-GlcNAcylation. In support of our hypothesis, a few of these protein sequences have already been reported to be involved in phosphorylation/O-GlcNAcylation crosstalk [e.g., in the eIF4 that plays a role in mRNA translation (24) and the paired amphipathic helix protein Sin3a that acts cooperatively with OGT to repress transcription (25,26)]. …”

O-GlcNAcylation Antagonizes Phosphorylation of CDH1 (CDC20 Homologue 1)

“…Spectra were labeled by pLabel with 20 ppm tolerance for fragment ions (27). GlcNAc specific reporters were also labeled (126.05495, 138.05495, 144.06551, 168.06552, 186.07608, 204.08665) (28).…”

Discovery of O-GlcNAc-6-phosphate Modified Proteins in Large-scale Phosphoproteomics Data

“…Data Analysis-The mass spectrometric data were processed essentially as described (6). The data processing with Mascot Distiller 2.4.2.0 (Matrix Science, London, UK) was slightly modified to account for the particular fragmentation behavior of O-GlcNAc-6-phosphate peptides.…”

“…For instance, the interplay between O-GlcNAcylation and phosphorylation modulates the stability and activity of p53 (5). However, recent data revealed the frequent co-occurrence of O-GlcNAc and phosphate at proximal sites (6), suggesting the reciprocal regulation by O-GlcNAcylation and phosphorylation may not be a very general mechanism. Moreover, it has also been found that the distribution of O-GlcNAc sites relative to phosphorylation sites is rather random and that the modification rates at sites detected with both modifications are almost equal, indicating that, on a global level, the substrate recognition of both pathways is not interconnected (7).…”

“…To interrogate such data systematically, we have recently developed a simple scoring scheme, termed Oscore, which automatically assesses tandem mass spectra for the presence and intensity of O-GlcNAc (HexNAc) diagnostic fragment ions and, in turn, allows ranking spectra according their probability of representing an O-GlcNAc peptide (15). A combined search strategy using the protein identification software Mascot and our Oscore algorithm enabled the identification of hundreds of O-GlcNAc peptides from large-scale proteome data (6).…”