Artificial Cysteine S-Glycosylation Induced by Per-O-Acetylated Unnatural Monosaccharides during Metabolic Glycan Labeling.

The unexpected, non-enzymatic S-glycosylation of cysteine residues in various proteins by per-O-acetylated monosaccharides is described. This artificial S-glycosylation greatly compromises the specificity and validity of metabolic glycan labeling in living cells by per-O-acetylated azido and alkynyl sugars, which has been overlooked in the field for decades. It is demonstrated that the use of unacetylated unnatural sugars can avoid the artifact formation and a corrected list of O-GlcNAcylated proteins and O-GlcNAc sites in HeLa cells has been assembled by using N-azidoacetylgalactosamine (GalNAz).

[1]  Xing Chen,et al.  Quantitative time-resolved chemoproteomics reveals that stable O-GlcNAc regulates box C/D snoRNP biogenesis , 2017, Proceedings of the National Academy of Sciences.

[2]  Xiaoyong Yang,et al.  Protein O-GlcNAcylation: emerging mechanisms and functions , 2017, Nature Reviews Molecular Cell Biology.

[3]  Ronghu Wu,et al.  Global and Site-Specific Analysis Revealing Unexpected and Extensive Protein S-GlcNAcylation in Human Cells. , 2017, Analytical chemistry.

[4]  M. Pratt,et al.  The Small Molecule 2-Azido-2-deoxy-glucose Is a Metabolic Chemical Reporter of O-GlcNAc Modifications in Mammalian Cells, Revealing an Unexpected Promiscuity of O-GlcNAc Transferase. , 2017, ACS chemical biology.

[5]  D. Vocadlo,et al.  Catalytic Promiscuity of O-GlcNAc Transferase Enables Unexpected Metabolic Engineering of Cytoplasmic Proteins with 2-Azido-2-deoxy-glucose. , 2017, ACS chemical biology.

[6]  A. Burlingame,et al.  Cysteine S-linked N-acetylglucosamine (S-GlcNAcylation), A New Post-translational Modification in Mammals* , 2016, Molecular & Cellular Proteomics.

[7]  C. Bertozzi,et al.  Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis , 2015, Nature Methods.

[8]  V. Piller,et al.  Changes in Metabolic Chemical Reporter Structure Yield a Selective Probe of O-GlcNAc Modification , 2014, Journal of the American Chemical Society.

[9]  B. Kuster,et al.  Proteome Wide Purification and Identification of O‑glcnac-modified Proteins Using Click Chemistry and Mass Spectrometry , 2022 .

[10]  H. Hang,et al.  Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1 , 2011, Proceedings of the National Academy of Sciences.

[11]  Jennifer J. Kohler,et al.  Metabolic cross-talk allows labeling of O-linked β-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway , 2011, Proceedings of the National Academy of Sciences.

[12]  Daniela C Dieterich,et al.  Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition. , 2010, Journal of the American Chemical Society.

[13]  Carolyn R Bertozzi,et al.  A chemical approach for identifying O-GlcNAc-modified proteins in cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  T. A. Fritz,et al.  Disaccharide uptake and priming in animal cells: inhibition of sialyl Lewis X by acetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol. , 1995, Proceedings of the National Academy of Sciences of the United States of America.