O-GlcNAcylation has gradually been recognized as a critically important protein post-translational modification in mammalian cells. Besides regulation of gene expression, its crosstalk with protein phosphorylation is vital for cell signaling. Despite its importance, comprehensive analysis of O-GlcNAcylation is extraordinarily challenging due to the low abundances of many O-GlcNAcylated proteins and the complexity of biological samples. Here, we developed a novel chemoenzymatic method based on a wild-type galactosyltransferase and uridine diphosphate galactose (UDP-Gal) for global and site-specific analysis of protein O-GlcNAcylation. This method integrates enzymatic reactions and hydrazide chemistry to enrich O-GlcNAcylated peptides. All reagents used are more easily accessible and cost-effective as compared to the engineered enzyme and click chemistry reagents. Biological triplicate experiments were performed to validate the effectiveness and the reproducibility of this method, and the results are comparable with the previous chemoenzymatic method using the engineered enzyme and click chemistry. Moreover, because of the promiscuity of the galactosyltransferase, 18 unique O-glucosylated peptides were identified on the EGF domain from nine proteins. Considering that effective and approachable methods are critical to advance glycoscience research, the current method without any sample restrictions can be widely applied for global analysis of protein O-GlcNAcylation in different samples.

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Glycoproteins with diverse glycans are essential to human cells, and subtle differences in glycan structures may result in entirely different functions. One typical example is proteins modified with O-linked β-N-acetylglucosamine (O-GlcNAc) and O-linked α-N-acetylgalactosamine (O-GalNAc) (the Tn antigen), in which the two glycans have very similar structures and identical chemical compositions, making them extraordinarily challenging to be distinguished. Here, we developed an effective method benefiting from selective enrichment and the enzymatic specificity to simultaneously identify and distinguish glycoproteins with O-GlcNAc and O-GalNAc. Metabolic labeling was combined with bioorthogonal chemistry for enriching glycoproteins modified with O-GlcNAc and O-GalNAc. Then, the enzymatic reaction with galactose oxidase was utilized to specifically oxidize O-GalNAc, but not O-GlcNAc, generating the different tags between glycopeptides with O-GlcNAc and O-GalNAc that can be easily distinguishable by mass spectrometry (MS). Among O-GlcNAcylated proteins commonly identified in three types of human cells, those related to transcription and RNA binding are highly enriched. Cell-specific features are also revealed. Among glycoproteins exclusively in Jurkat cells, those involved in human T-lymphotropic virus type 1 (HTLV-1) infection are overrepresented, which is consistent with the cell line source and suggests that protein O-GlcNAcylation participated in the response to the virus infection. Furthermore, glycoproteins with the Tn antigen have different subcellular distributions in different cells, which may be attributed to the distinct mechanisms for the formation of protein O-GalNAcylation.

Protein O-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 O-GlcNAcylation can advance our understanding of this important modification. However, it is extraordinarily challenging because normally O-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 O-GlcNAcylation. The boosting approach using the TMT labeling dramatically enhances the detection of co-translational glycopeptides with low abundance when enriched O-GlcNAcylated peptides from cells with a much longer labeling time was used as a boosting sample. More than 180 co-translational O-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 O-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 O-GlcNAcylation, which is very useful to advance our understanding of this important modification.

Protein O-GlcNAcylation refers to the covalent binding of a single N-acetylglucosamine (GlcNAc) to the serine or threonine residue. This modification primarily occurs on proteins in the nucleus and the cytosol,...