Combining Selective Enrichment and a Boosting Approach to Globally and Site-Specifically Characterize Protein Co-translational <i>O</i>‑GlcNAcylation

Protein <i>O</i>-GlcNAcylation plays extremely important roles in mammalian cells, regulating signal transduction and gene expression. This modification can happen during protein translation, and systematic and site-specific analysis of protein co-translational <i>O</i>-GlcNAcylation can advance our understanding of this important modification. However, it is extraordinarily challenging because normally <i>O</i>-GlcNAcylated proteins are very low abundant and the abundances of co-translational ones are even much lower. Here, we developed a method integrating selective enrichment, a boosting approach, and multiplexed proteomics to globally and site-specifically characterize protein co-translational <i>O</i>-GlcNAcylation. The boosting approach using the TMT labeling dramatically enhances the detection of co-translational glycopeptides with low abundance when enriched <i>O</i>-GlcNAcylated peptides from cells with a much longer labeling time was used as a boosting sample. More than 180 co-translational <i>O</i>-GlcNAcylated proteins were site-specifically identified. Further analyses revealed that among co-translational glycoproteins, those related to DNA binding and transcription are highly overrepresented using the total identified <i>O</i>-GlcNAcylated proteins in the same cells as the background. Compared with the glycosylation sites on all glycoproteins, co-translational sites have different local structures and adjacent amino acid residues. Overall, an integrative method was developed to identify protein co-translational <i>O</i>-GlcNAcylation, which is very useful to advance our understanding of this important modification.