Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries

Citation:

Mehta AY, Veeraiah RKH, Dutta S, Goth CK, Hanes MS, Gao C, Stavenhagen K, Kardish R, Matsumoto Y, Heimburg-Molinaro J, et al. Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries. Cell Chemical Biology. 2020;27 (9) :1207-1219.

Abstract:

Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans.

Last updated on 10/14/2020