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Screening Carbohydrate Libraries for Protein Interactions Using the Direct ESI-MS Assay. Applications to Libraries of Unknown Concentration

  • Research Article
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Journal of The American Society for Mass Spectrometry

Abstract

A semiquantitative electrospray ionization mass spectrometry (ESI-MS) binding assay suitable for analyzing mixtures of oligosaccharides, at unknown concentrations, for interactions with target proteins is described. The assay relies on the differences in the ratio of the relative abundances of the ligand-bound and free protein ions measured by ESI-MS at two or more initial protein concentrations to distinguish low affinity (≤103 M–1) ligands from moderate and high affinity (>105 M–1) ligands present in the library and to rank their affinities. Control experiments were performed on solutions of a single chain antibody and a mixture of synthetic oligosaccharides, with known affinities, in the absence and presence of a 40-component carbohydrate library to demonstrate the implementation and reliability of the assay. The application of the assay for screening natural libraries of carbohydrates against proteins is also demonstrated using mixtures of human milk oligosaccharides, isolated from breast milk, and fragments of a bacterial toxin and human galectin 3.

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Acknowledgments

The authors acknowledge the Alberta Glycomics Center and the Natural Sciences and Engineering Research Council of Canada for funding, and Professor D. Bundle (University of Alberta) for generously providing some of the oligosaccharides used in this study, Professor K. Ng (University of Calgary) for the gift of TcdB fragment, and Professor C. Cairo (University of Alberta) for providing the fragment of human galectin 3.

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Author notes

  1. Amr El-Hawiet

    Present address: Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt

Authors and Affiliations

  1. Alberta Glycomics Center and Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada

    Elena N. Kitova, Amr El-Hawiet & John S. Klassen

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  1. Elena N. Kitova
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  2. Amr El-Hawiet
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  3. John S. Klassen
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Correspondence to John S. Klassen.

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Kitova, E.N., El-Hawiet, A. & Klassen, J.S. Screening Carbohydrate Libraries for Protein Interactions Using the Direct ESI-MS Assay. Applications to Libraries of Unknown Concentration. J. Am. Soc. Mass Spectrom. 25, 1908–1916 (2014). https://doi.org/10.1007/s13361-014-0964-2

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  • DOI: https://doi.org/10.1007/s13361-014-0964-2

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