Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting

Biochemistry
Narek DarabedianMatthew R Pratt

Abstract

The dynamic modification of intracellular proteins by O-linked β -N-acetylglucosamine (O-GlcNAcylation) plays critical roles in many cellular processes. Although various methods have been developed for O-GlcNAc detection, there are few techniques for monitoring glycosylation stoichiometry and state (i.e., mono-, di-, etc., O-GlcNAcylated). Measuring the levels of O-GlcNAcylation on a given substrate protein is important for understanding the biology of this critical modification and for prioritizing substrates for functional studies. One powerful solution to this limitation involves the chemoenzymatic installation of polyethylene glycol polymers of defined molecular mass onto O-GlcNAcylated proteins. These "mass tags" produce shifts in protein migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that can be detected by Western blotting. Broad adoption of this method by the scientific community has been limited, however, by a lack of commercially available reagents and well-defined protein standards. Here, we develop a "click chemistry" approach to this method using entirely commercial reagents and confirm the accuracy of the approach using a semisynthetic O-GlcNAcylated protein. Our studies esta...Continue Reading

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Citations

Sep 16, 2020·Nature Chemical Biology·Nichole J PedowitzMatthew R Pratt
Sep 18, 2020·Molecular & Cellular Proteomics : MCP·Jason C Maynard, Robert J Chalkley
Jan 9, 2021·Chemical Reviews·Junfeng MaGerald W Hart
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Feb 16, 2020·Cell·Christopher G Parker, Matthew R Pratt
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Oct 31, 2020·Current Opinion in Chemical Biology·Anna CioceBenjamin Schumann
Feb 16, 2021·Molecular & Cellular Proteomics : MCP·Jason C Maynard, Robert J Chalkley
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Jun 9, 2021·Chembiochem : a European Journal of Chemical Biology·Eun Ju Kim
Jul 13, 2021·Frontiers in Chemistry·Qiang Zhu, Wen Yi
Aug 3, 2021·Chemical Society Reviews·Abhijit SahaAlberto Fernández-Tejada
Oct 19, 2019·Analytical Chemistry·Suttipong SuttapitugsakulRonghu Wu
Jun 12, 2020·RSC Chemical Biology·Andrii Gorelik, Daan M F van Aalten
Dec 23, 2021·Nature Chemical Biology·Charlie Fehl, John A Hanover

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