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GlycoProfile™ Azido Sugars: Flag Phosphine Technology

BioFiles 2007, 2.1, 16.

BioFiles 2007, 2.1, 16.

Advancing Analysis of Glycoprotein Processing for both Intra and Extra-cellular Evaluation

Many intracellular processing events are disrupted environmentally or are the result of genomic abnormalities (congenital disorders of glycosylation; CDG) and result in disease states. Multiple studies have evaluated the roles of glycoproteins and proteoglycans in tumor metastasis, angiogenesis, inflammatory cell migration, lymphocyte homeostasis, and congenital disorders of glycosylation. Stepwise analysis of the intracellular and surface-displayed sugars provides researchers a more complete picture of the process.

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Bioorthogonal Chemical Reporters

While changes in N and O-linked protein glycosylation are known to correlate with disease states, those changes are difficult to monitor in a physiological setting because of a lack of experimental tools. Sigma, in collaboration with the research community, has developed tools for profiling N- and O-linked glycoproteins by labeling cellular glycans using an alternative metabolic-system approach that works both in vitro and in vivo.1-5 Non-natural azido-containing monosaccharides (see Figure 1) that are bioorthogonal chemical reporters are introduced into a cell and incorporated into glycan structures through endogenous glycosylation processes (see Figure 2).

Figure 1. Azido sugars for incorporation into glycan structures.

Figure 2. Possible sites of azido-sugar incorporation in simple and complex N-linked glycan structures.

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Incorporation of Azido Sugars in Carbohydrate Structures

Cells metabolize the azidosugars using glycosyltransferases and express the sugars on the terminus of a glycan chain both intracellularly and on the cell surface, leaving the azido group unreacted. The azidosugars can also be incorporated into glycans via the sialic acid metabolic pathway. A selective phosphine probe containing a detection epitope such as FLAG® is applied to the cellular extracts containing the azidoglycans. The phosphine group selectively reacts via Staudinger ligation with the displayed azido group, resulting in an epitope tag covalently attached to the glycan (see Figure 3).

Figure 3. Profiling N-type glycoproteins by metabolic labeling with an azido GalNAc analog (GalNAz) followed by Staudinger ligation with a phosphine probe(phosphine-FLAG). R and R’ are oligosaccharide elaborations from the core GalNAc residue.

Although non-natural molecules, both the azido and phosphine moieties are tolerated during cell proliferation. The bound epitope peptide is then detected by using FLAG-specific antibody. This approach permits the analysis of pathways that are regulated by particular glycan post-translational modifications as well as the monitoring of the intracellular glycosylation process itself. Metabolic labeling with bioorthogonal chemical reporters such as azidosugars followed by Staudinger ligation provides a unique mechanism for proteomic analysis of this post-translational modification and for identifying glycoprotein fingerprints associated with disease.

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  1. Cell surface engineering by a modified Staudinger reaction. Saxon, E. and Bertozzi, C.R., Science , 287, 2007 (2000).
  2. Chemical remodelling of cell surfaces in living animals. Prescher, J.A., Dube, D.H., and Bertozzi, C.R., Nature, 430, 873 (2004).
  3. A chemical approach for identifying O-GlcNAc-modified proteins in cells. Vocadlo, D.J., Hang, H.C., Kim, E.J., Hanover, J.A., Bertozzi, C.R., Proc. Natl. Acad. Sci. USA, 100, 9116 (2004).
  4. A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation, Hang H.C., Yu, C., Kato, D.L., Bertozzi, C.R., Proc. Natl. Acad. Sci. USA, 100, 14846 (2003).
  5. Probing mucin-type O-linked glycosylation in living animals. Dube, D.H., Prescher J.A., Quang C.N., and Bertozzi, C.R. Proc. Natl. Acad. Sci. USA, 103, 4819 (2006)

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