Quantitative Profiling of Protein O‑GlcNAcylation Sites by an Isotope-Tagged Cleavable Linker

Large-scale quantification of protein
O-linked β-N-acetylglucosamine (O-GlcNAc) modification
in a site-specific
manner remains a key challenge in studying O-GlcNAc biology. Herein,
we developed an isotope-tagged cleavable linker (isoTCL) strategy,
which enabled isotopic labeling of O-GlcNAc through bioorthogonal
conjugation of affinity tags. We demonstrated the application of the
isoTCL in mapping and quantification of O-GlcNAcylation sites in HeLa
cells. Furthermore, we investigated the O-GlcNAcylation sensitivity
to the sugar donor by quantifying the levels of modification under
different concentrations of the O-GlcNAc labeling probe in a site-specific
manner. In addition, we applied isoTCL to compare the O-GlcNAcylation
stoichiometry levels of more than 100 modification sites between placenta
samples from male and female mice and confirmed site-specifically
that female placenta has a higher O-GlcNAcylation than its male counterpart.
The isoTCL platform provides a powerful tool for quantitative profiling
of O-GlcNAc modification.