A significant fraction of the proteome is decorated with glycans and many of the resulting protein glycoconjugates serve as molecular binding targets for small molecules. The glycosylation states of glycoproteins can profoundly influence these binding events, leading to important biological consequences, including altering the efficacy of small molecule therapeutics.  The presence of the glycan can directly camouflage a protein binding surface or it can indirectly alter protein conformation. To date, however, there are only an isolated handful of examples describing how glycosylation can modulate the binding of small molecules to glycoproteins. Thus, approaches to expedite the identification of glycoproteins that experience changes in ligandability are greatly needed, especially those that can facilitate this discovery in live cells. Using fragment-based photoaffinity probes coupled with quantitative mass spectrometry-based proteomics, we systematically identify ~250 glycoproteins that experience changes in ligandability in live cells upon the loss of N-linked glycosylation or heparan sulfate modification. Using a series of biochemical experiments, we validate and confirm the observed changes in glycosylation-dependent ligandability. Furthermore, we identify the corresponding N-linked glycosites that engage the fragments with the loss of glycosylation. Our study reveals that biosynthetic glycosylation greatly influences the ligandable proteome, and the resulting fragment molecules have the potential to be advanced toward more selective ligands that can discriminate against differentially glycosylated protein glycoconjugates.

Mapping glycosylation-dependent changes in glycoprotein ligandability across the proteome

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