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

  • Elena N. Kitova
  • Amr El-Hawiet
  • John S. Klassen
Research Article

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.

Graphical Abstract

Keywords

Carbohydrate Library screening Protein interactions 

Notes

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.

Supplementary material

13361_2014_964_MOESM1_ESM.docx (551 kb)
ESM 1(DOCX 551 kb)

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Copyright information

© American Society for Mass Spectrometry 2014

Authors and Affiliations

  • Elena N. Kitova
    • 1
  • Amr El-Hawiet
    • 1
    • 2
  • John S. Klassen
    • 1
  1. 1.Alberta Glycomics Center and Department of ChemistryUniversity of AlbertaEdmontonCanada
  2. 2.Faculty of PharmacyUniversity of AlexandriaAlexandriaEgypt

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