Measuring O-glycosylation: A quick game of tagFunctional Glycomics (09 September 2010) | doi:10.1038/fg.2010.28
A simple new method uses mass tags to measure O-glycosylation stoichiometries and dynamics on unpurified, endogenous proteins.
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O-GlcNAc glycosylation, the addition of N-acetyl-D-glucosamine to intracellular proteins, plays critical roles in diverse biological processes. However, detecting and studying this modification is difficult, traditionally requiring the use of purified proteins, radiolabels, or site-specific antibodies. Reporting in Nature Chemical Biology, Linda Hsieh-Wilson and colleagues sweep away these obstacles with a new method that uses mass tags to measure the stoichiometry and dynamics of O-GlcNAc glycosylation on unpurified proteins from cell and tissue lysates. Using this method they examine the dynamic interplay between glycosylation and phosphorylation on individual proteins.
At the heart of the approach is the attachment of a single polyethylene glycol (PEG) 'mass tag' of known molecular weight to individual O-GlcNAc moieties on mixtures of proteins from cell lysates. The authors anticipated that each attached PEG tag, labeling a single O-GlcNAc site, would slow a protein's migration during gel electrophoresis by a defined amount, allowing glycosylation stoichiometries to be determined simply by immunoblotting with protein-specific antibodies and quantifying relative band intensities. To accomplish this, a modified galactosyltransferase was used to transfer a keto-functionalized non-natural galactose residue to GlcNAc, enabling the further bioorthogonal coupling of an aminooxy-functionalized PEG. The authors validated the method using simple methods and known standards and demonstrated that it is sensitive enough to detect glycosylation of as little as 0.8% of an individual protein in a mixture.
A major potential application of this approach is comparison of endogenous O-glycosylation across different types of tissues, disease states, or experimental treatments. For instance, mass tagging of proteins from rat tissues revealed that O-glycosylation levels can vary significantly across different proteins in a single tissue, and on the same protein in different tissues. As one example, 45% of the transcription factor CREB was glycosylated in rat brain compared with 32% in liver. This highly reproducible result suggests tight regulatory control of physiological O-GlcNAc levels. The authors also measured dynamic cycling of O-glycosylation over time following changes in UDP-GlcNAc synthesis, and found that existing O-GlcNAc moieties can slow O-glycosylation of other residues on the same protein.
The ability to examine the dynamic interplay between glycosylation and phosphorylation on a protein by combining mass tagging with phospho-specific immunoblotting is another breakthrough of this method. Whereas glycosylation and phosphorylation of CREB—in response to specific stimuli—were independent, on the transcription factor MeCP2 the modifications showed a surprising 'reverse yin–yang' relationship, wherein glycosylation promoted phosphorylation and vice–versa. Importantly, this interplay was only revealed by examining specific glycosylated subpools of the protein, which would not have been possible without the mass tagging technique.
The mass tagging approach opens the door for O-GlcNAc stoichiometries and dynamics to be measured on any endogenous protein that can be detected on a western blot, and in the future should provide important insights into the dynamic interplay between O-GlcNAc glycosylation and other post-translational modifications.
Original research paper
- Rexach, J. E., Rogers, C. J., Yu, S-H., Tao, J., Sun, Y. E., and Hsieh-Wilson, L. C. Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags. Nat. Chem. Biol. 6, 645–651 (2010) doi:10.1038/nchembio.412 | Article