Cell Chemical Biology
Volume 27, Issue 9, 17 September 2020, Pages 1207-1219.e9
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Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries

https://doi.org/10.1016/j.chembiol.2020.06.007Get rights and content
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Highlights

  • The Glyco-SPOT synthesis technique was developed to generate glycopeptide libraries

  • Libraries consisted of variable length glycopeptides with O-, N-, and S-linked glycans

  • Used for microarray screening, enzyme specificity, affinity, mass spectrometry

  • Technique opens up more abilities to study biology of glycopeptides

Summary

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.

Keywords

glycopeptide
glycan
microarray
mass spectrometry
glycosyltransferase
glycopeptide library
CTD110.6
S-GlcNAcylation
ST6GalNAc1
TSynthase
GCNT1

Cited by (0)

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Present address: Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, Copenhagen N 2200, Denmark

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Present address: Visterra Inc. 275 2nd Avenue, Waltham, MA 02451, USA

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Present address: Scienion US, 2640 West Medtronic Way, Tempe, AZ 85281, USA

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