In our laboratories, we have optimized the synthesis of O-glycopepties for the production of O-glycopeptide microarrays, in order dramatically simplify the purification requirements for each synthetic target. Peptide microarrays have evolved during the last decade as a powerful tool for high-throughput screening of a host of biological events. We and others have shown that microarray format can be harnessed to display up to hundreds of different synthetic O-glycopeptides, anchored in a covalent fashion, for seromic profiling. It combines parallel and automated Fmoc-SP glycopeptides synthesis at a scale of 2 μmo. This entails capping of unreacted N-terminal amines after each coupling cycle, so that ultimately, only full-length target structures retain the free N-terminal amine. Next, the glycopeptides are used directly for the construction of glycopeptide microarrays. The fact that only full-length glycopeptides have an N-terminal amine enables an on-chip purification technique as these amine-functionalized glycopeptides can be covalently captured by amine reactive glass slides (NHS-ester present throughout the surface, Figure). The slide support we apply, by virtue of its hydrogel surface, has proven to be particularly suited for sera screening giving very low unspecific binding and background in the off-feature areas. Moreover, enzymatic elongation of glycan haptens is also well tolerated, thus giving access to a multilayered screening platform.
- Blixt O, Westerlind U. Arraying the post-translational glycoproteome (PTG). Curr Opin Chem Biol. 2014 Feb;18:62-9.
- Blixt O, Cló E. Synthesis of O-glycopeptides and construction of glycopeptide microarrays. Methods Mol Biol. (2013), 1047:201-14.
- Blixt O, Cló E, Nudelman AS, Sørensen KK, Clausen T, Wandall HH, Livingston PO, Clausen H, Jensen KJ. A high-throughput O-glycopeptide discovery platform for seromic profiling. (2010) J Proteome Res. 9(10):5250-61.