A quantitative, high-throughput method identifies protein\u2013glycan interactions via mass spectrometry

Pavel I Kitov,Matthew S Macauley,Elena N Kitova,Carmanah D Hunter,Ling Han,Emily Rodrigues,John S Klassen,Jaesoo Jung,Zhixiong Li,Christopher W Cairo

doi:10.1038/s42003-019-0507-2

Pavel I Kitov, Matthew S Macauley + Show 8 more

Open Access

https://doi.org/10.1038/s42003-019-0507-2

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Abstract

Glycan binding by glycan-binding proteins and processing by carbohydrate-active enzymes is implicated in physiological and pathophysiological processes. Comprehensive mapping of glycan interactions is essential to understanding of glycan-mediated biology and can guide the development of new diagnostics and therapeutics. Here, we introduce the competitive universal proxy receptor assay (CUPRA), which combines electrospray ionization mass spectrometry, competitive binding and heterobifunctional glycan-based ligands to give a quantitative high-throughput method for screening glycan libraries against glycan-binding and glycan-processing proteins. Application of the assay to human (siglec-2), plant (Sambucus nigra and Maackia amurensis lectins) and bacterial (cholera toxin, and family 51 carbohydrate binding module) proteins allowed for the identification of ligands with affinities (Kd) ≤ 1 mM. The assay is unprecedentedly versatile and can be applied to natural libraries and, when implemented in a time-resolved manner, provides a quantitative measure of the activities and substrate specificity of carbohydrate-active enzymes.

Highlights

Glycan binding by glycan-binding proteins and processing by carbohydrate-active enzymes is implicated in physiological and pathophysiological processes
We present the competitive universal proxy receptor assay (CUPRA), a method that employs a library of heterobifunctional compounds, consisting of oligosaccharides linked to a common affinity tag, and electrospray ionization mass spectrometry (ESI-MS) to simultaneously measure coupled binding equilibria involving the library, a universal proxy protein receptor (UniPproxy), which binds all components of the library through the affinity tag, and target glycan-binding proteins (GBPs) (Fig. 1a)
We introduced the CUPRA linker, which consists of an affinity tag based on the sulfonamide group and a short PEG dipeptide linker, to the reducing end of free oligosaccharides with an N-glycosidic linkage using established chemistry (Fig. 1b and Supplementary Fig. 1)

Summary

Introduction

Glycan binding by glycan-binding proteins and processing by carbohydrate-active enzymes is implicated in physiological and pathophysiological processes. We introduce the competitive universal proxy receptor assay (CUPRA), which combines electrospray ionization mass spectrometry, competitive binding and heterobifunctional glycan-based ligands to give a quantitative high-throughput method for screening glycan libraries against glycan-binding and glycan-processing proteins. In which oligosaccharides are immobilized through a linker on a solid surface, is the dominant technology for high-throughput screening of oligosaccharides libraries, both synthetic and natural[4]. Despite their extensive use for establishing glycan-binding specificities of GBPs, glycan array screening has a number of well-known limitations. An alternative to glycan microarrays is catch-and-release electrospray ionization mass spectrometry (ESI-MS), a label- and immobilization-free assay capable of simultaneously screening hundreds of oligosaccharides against GBPs7. Analysis of the magnitude of the changes, using a binding model that takes into account all possible interactions between the library components and GBP and UniPproxy, provides the affinities of the GBP ligands that are detected

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