Mass Spectrometry-Based Shotgun Glycomics for Discovery of Natural Ligands of Glycan-Binding Proteins

Anal Chem. 2020 Oct 20;92(20):14012-14020. doi: 10.1021/acs.analchem.0c02931. Epub 2020 Oct 6.


Glycans attached to lipids and membrane-bound and secreted proteins and peptides mediate many important physiological and pathophysiological processes through interactions with glycan-binding proteins (GBPs). However, uncovering functional glycan ligands is challenging due to the large number of naturally occurring glycan structures, the limited availability of glycans in their purified form, the low affinities of GBP-glycan interactions, and limitations in existing binding assays. This work explores the application of catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) for screening libraries of N-glycans derived from natural sources. The assay was tested by screening a small-defined library of complex N-glycans at equimolar concentrations against plant and human GBPs with known specificities for either α2-3- or α2-6-linked sialosides, with affinities in the millimolar to micromolar range. Validation experiments, performed in negative ion mode, revealed that bound N-glycan ligands are readily released, as intact deprotonated ions, from GBPs in the gas phase using collision-induced dissociation. Moreover, the relative abundances of the released ligands closely match their solution affinities. The results obtained for a natural N-glycan library produced from cultured immune cells serve to highlight the ease with which CaR-ESI-MS can screen complex mixtures of N-glycans for interactions. Additionally, scaling the relative abundances of released glycan ligands according to their relative abundances in solution, as determined by hydrophilic interaction-ultrahigh-performance liquid chromatography of the fluorescently labeled library, allows the relative affinities of glycan ligands to be ranked.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agglutinins / chemistry
  • Agglutinins / metabolism
  • Chromatography, High Pressure Liquid
  • Glycomics / methods*
  • Humans
  • Ligands
  • Plant Lectins / chemistry
  • Plant Lectins / metabolism
  • Polysaccharides / analysis*
  • Polysaccharides / metabolism
  • Protein Binding
  • Sambucus nigra / metabolism
  • Sialic Acid Binding Ig-like Lectin 2 / chemistry
  • Sialic Acid Binding Ig-like Lectin 2 / metabolism
  • Spectrometry, Mass, Electrospray Ionization*


  • Agglutinins
  • CD22 protein, human
  • Ligands
  • Plant Lectins
  • Polysaccharides
  • Sialic Acid Binding Ig-like Lectin 2