Simultaneously Identifying and Distinguishing Glycoproteins with O-GlcNAc and O-GalNAc (the Tn Antigen) in Human Cancer Cells

Abstract

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.

Combining Selective Enrichment and a Boosting Approach to Globally and Site-Specifically Characterize Protein Co-translational O-GlcNAcylation

“…18 Sites with ModScore >13 (P < 0.05) were considered as confidently localized, and only the confidently localized sites were used for site-specific analysis. 19,20 To remove S-glycosylation sites, we have applied very stringent criteria to make sure that only confidently identified O-glycosylation sites were included in the data set. The detailed filtering steps are the same with our previous publications.…”

“…The detailed filtering steps are the same with our previous publications. 6,20 The isotopic correction parameters provided by Thermo were used to calibrate the TMT reporter ion intensities before data analysis. For the two TMT channels for the samples of co-translational O-GlcNAcylation, the S/N ratio in at least one sample has to be >5 for confident annotation of co-translational O-GlcNAcylated peptides.…”

“…ModScore was used to determine the confidence of the glycosylation site localization . Sites with ModScore >13 ( P < 0.05) were considered as confidently localized, and only the confidently localized sites were used for site-specific analysis. , To remove S -glycosylation sites, we have applied very stringent criteria to make sure that only confidently identified O -glycosylation sites were included in the data set. The detailed filtering steps are the same with our previous publications. , The isotopic correction parameters provided by Thermo were used to calibrate the TMT reporter ion intensities before data analysis.…”

“…Sites with ModScore >13 ( P < 0.05) were considered as confidently localized, and only the confidently localized sites were used for site-specific analysis. , To remove S -glycosylation sites, we have applied very stringent criteria to make sure that only confidently identified O -glycosylation sites were included in the data set. The detailed filtering steps are the same with our previous publications. , The isotopic correction parameters provided by Thermo were used to calibrate the TMT reporter ion intensities before data analysis. For the two TMT channels for the samples of co-translational O -GlcNAcylation, the S / N ratio in at least one sample has to be >5 for confident annotation of co-translational O -GlcNAcylated peptides.…”

Tunable Approach to C-Linked Analogs of Glycosamines

“…2-Amino-2-deoxy-sugars and their N- and O -glycoconjugates are ubiquitous key biomolecules that are involved in numerous fundamental biological events . Typical examples contain d -glucosamine, − d -galactosamine, , d -allosamine, and d -mannosamine. , …”