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Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries.

Cell chemical biology (2020-07-02)
Akul Y Mehta, Ravi Kumar H Veeraiah, Sucharita Dutta, Christoffer K Goth, Melinda S Hanes, Chao Gao, Kathrin Stavenhagen, Robert Kardish, Yasuyuki Matsumoto, Jamie Heimburg-Molinaro, Michael Boyce, Nicola L B Pohl, Richard D Cummings
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.

MATERIALS
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