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Optimization of chemoenzymatic mass-tagging by strain-promoted cycloaddition (SPAAC) for the determination of O-GlcNAc stoichiometry by Western blotting

Darabedian, Narek and Thompson, John W. and Chuh, Kelly N. and Hsieh-Wilson, Linda C. and Pratt, Matthew R. (2018) Optimization of chemoenzymatic mass-tagging by strain-promoted cycloaddition (SPAAC) for the determination of O-GlcNAc stoichiometry by Western blotting. Biochemistry, 57 (40). pp. 5769-5774. ISSN 0006-2960.

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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 establish a new, expedited experimental workflow and standardized methods that can be readily utilized by non-experts to quantify the O-GlcNAc stoichiometry and state on endogenous proteins in any cell or tissue lysate.

Item Type:Article
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URLURL TypeDescription Information
Thompson, John W.0000-0003-0061-4996
Hsieh-Wilson, Linda C.0000-0001-5661-1714
Pratt, Matthew R.0000-0003-3205-5615
Additional Information:© 2018 American Chemical Society. Received: June 12, 2018; Revised: August 16, 2018; Published: August 31, 2018. This research was supported by the National Science Foundation (Grant CHE-1506503 to M.R.P.), the National Institutes of Health (Grants R01GM114537 and R01GM125939 to M.R.P., R01GM084724 and R01AG060540-13 to L.C.H.-W., and T32GM008042, T32GM007616, and F30AG055314 to J.W.T.), and the UCLA-Caltech Medical Scientist Training Program (J.W.T.). The authors also thank Dr. P. Qasba (National Cancer Institute at Frederick, Frederick, MD) for generously providing the GalT(Y289L) construct. The authors declare no competing financial interest.
Funding AgencyGrant Number
NIH Predoctoral FellowshipT32GM008042
NIH Predoctoral FellowshipT32GM007616
NIH Postdoctoral FellowshipF30AG055314
UCLA-Caltech Medical Scientist Training ProgramUNSPECIFIED
Record Number:CaltechAUTHORS:20180904-103320708
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Official Citation:Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting. Narek Darabedian, John W. Thompson, Kelly N. Chuh, Linda C. Hsieh-Wilson, and Matthew R. Pratt. Biochemistry 2018 57 (40), 5769-5774 DOI: 10.1021/acs.biochem.8b00648
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89356
Deposited By: Tony Diaz
Deposited On:04 Sep 2018 17:42
Last Modified:10 Oct 2018 17:04

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